Proteomic Analysis Of The Rat Model Of Ketamine Associated-cystitis And Verification Of Transgelin

BACKGROUND&OBJECTIVEKetamine is a derivative of phencyclidine and act as a dissociative anesthetic. However, its use as a recreational drug is on the increase among young adults. Several clinical studies have reported that long-term ketamine abuse can affect urinary system, causing lower urinary tract syndrome, such as frequency, urgency, suprapubic discomfort and times hematuria. Cystoscopy findings and histological changes of the bladder biopsies of the ketamine abusers are similar to those with interstitial cystitis. Severe cases usually developed into small bladder capacity and irreversible dysfunction of urinary system. However, how ketamine abuse lead to these interstitial cystitis-like symptoms and histological changes is not clear. It was suggested by Chu et al. that ketamine and its metabolites may have a direct toxic effect on the bladder epithelial, causing chronic submucosal and detrusor muscle inflammatory response, leading to IC-like symptoms and contracture of bladder.Our previous study had successfully created a rat model of ketamine associated cystitis. After16weeks of ketamine treatment, rats developed severe urinary frequency and hematuria. Histopathological study showed thicker submucosal layer with fibrosis and inflammation in the bladder wall. In this study, we hypothesize that identification of proteins in the bladder through proteomic analysis may lead to novel insights into the underlying mechanisms. The different expressions of the tissue proteins in the bladder of rat were compared between normal saline group and ketamine-treated group by using two-dimensional difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and validated by immunohistochemistry study and western blot analysis.METHODS1. Establishment of rat model of ketamine associated-cystitis and pathology assessment.Twenty-four2-month-old male Sprague-Dawley (SD) rats weighing180-190g were purchased from the Animal Center of Southern Medical University (SMU). Rats were randomLy assigned to control, normal saline (NS), low-dose (10mg.kg-1, LK) and high-dose (50mg.kg-1, HK) groups with6in each; these doses being based on preliminary experiments. The two experimental groups were given ketamine hydrochloride i.p. daily at9.am, dissolved in500μl saline. The NS group rats were injected i.p. with the vehicle the same time, while the6rats in control group were untreated.The rats were anesthetize by chloralic hydras, and the bladders were excised and divided into three midsagittal portions. The first part was fixed in4%paraformaldehyd for histological examination (H&E staining, Masson staining),the second part was used for2D-DIGE protein analysis, and the third part was stored in liquid N2for western blot analysis. All animal care and procedures were in accordance with China and Nanfang Hospital policies for health and well-being of animals.2. Sample preparation for DIGE analysisTissue samples from HK group and NS group were used for2D-DIGE protein analysis. After2-DE, images of protein spots in the gels were scanned and captured by a UMAX PowerLook1100scanner. The obtained images were analyzed using PDQuest7.1.0software. The ratio of differentially expressed protein abundance which increased or decreased more than1.5-fold were considered significant changes (P<0.05).3. MALDI-TOF/MS analysis and database searchingThe selected peptide mixture was measured on a Voyager-DE STR MALDI-TOF mass spectrometer. The peptide mass fingerprint (PMF) data and MS data were combined and the combined data set was submitted to MASCOT for protein identification. The searching parameters were set as follows:rattus norvegicus, trypsin cleavage (one missed cleavage allowed), and Carbamidomethyl (C), Oxidation (M) as fixed modification.4. Phosphoprotein purification and verification of selected proteinsPhosphoprotein purification columns were used to purify phosphoprotein and non-phosphoprotein samples of blank control, NS, LK, and HK groups. Immunohistochemistry and western blot were performed in all groups.5. Phosphorylated protein for2-DE analysis and MALDI-TOF/MS analysisPhosphoprotein of HK group and NS group were used for2-DE analysis. After2-DE, images of protein spots in the gels were scanned and captured by a UMAX PowerLook1100scanner. The obtained images were analyzed using PDQuest7.1.0software. The ratio of differentially expressed protein abundance which increased or decreased more than1.5-fold were considered significant changes (P<0.05). The selected peptide mixture was measured on a Voyager-DE STR MALDI-TOF mass spectrometer. The peptide mass fingerprint (PMF) data and MS data were combined and the combined data set was submitted to MASCOT for protein identification.RESULTS1. After12weeks of ketamine treatment, rats in both LK and HK groups showed significant increase in micturition frequency when compared to NS group. After16weeks of ketamine treatment, three of six rats in HK group developed haematuria. H&E staining showed hyperplastic epithelium, inflammatory cells infiltration in the HK-treated rat bladders. Masson staining showed hyperplasia of fibre tissue in submucosal and smooth muscle layers in both LK and HK-treated rat bladders. 2. Approximately1,200protein spots were detected on each gel by2D-DIGE.30altered expressions were chosen between NS and HK-treated group. Compared to NS-treated rat bladder (Cy3, green),14up-regulated spots and16down-regulated spots in the HK-treated rat bladder (Cy5, red), with fold-changes ranging from+4.32to+11.74and-4.17to-10.98(p<0.05), were chosen by PDQuest7.1.0software.3. Eventually,29of these30protein spots were successfully identified by MALDI-TOF/MS, and by subsequent comparative sequence searches in the Mascot database. Among these,14up-regulated proteins were as follows:T-kininogen, Plastin, Putative protein, ATP-citrate synthas, mitochondrial Aldehyde dehydrogenase, Actin, Heat shock protein beta-6, Transgelin, Protein S100-A6and Eukaryotic translation initiation factor5A-1.16down-regulated proteins were as follows:Endoplasmin, PCI domain-containing protein2, Serum albumin, Keratin, type Ⅰ cytoskeletal, Protein disulfide-isomerase A3, Actin, Phospholysine phosphohistidine inorganic pyrophosphate phosphatase, Lamin-A/C, Aldosereductase, Dihydrolipoyllysine-residue, succinyltransferase component of2-oxoglutarate dehydrogenase complex, Fructose-1,6-bisphosphatase1, Phosphatidylinositol transfer protein alpha isoform and Transgelin. Among these, the up-regulated protein spots A11and A12, as well as the down-regulated spots B15and B16were all identified as transgelin. So far, Transgelin was the unique protein of the identified proteins that was related to investigations of cystitis.4. Immunohistochemical staining and western blot analysis showed that the expression of total transgelin had no significant difference between groups. However, the expression of phosphorylated transgelin in LK and HK groups was increased, while the non-phosphorylated transgelin was decreased when compared to the NS group.5. Approximately840phosphorylated protein spots were detected on each gel by2-DE.8altered expressions were identified between NS and HK-treated group. One of the phosphorylated protein spots was successfully identified by MALDI-TOF/MS as Transgelin. CONCLUSION1. Ketamine associated rat model was established successfully. In our study, long-term ketamine abuse in rat induced severe urinary frequency, and the pathological analysis revealed abnormal epithelium and inflammatory cells infiltration in submucosal layer. All these phenomena were similar to those patients and mice after long term ketamine abuse or administration.2.14up-regulated and15down-regulated proteins were successfully identified by2D-DIGE followed by MALDI-TOF/MS eventually. Protein Transgelin was chosen for further investigated.3. We hypothesized that long-term ketamine treatment can active the phosphorylation of transgelin in the bladder wall, inhibiting its binding with actin, and thus impair the regulation of smooth muscle and affect the contractility of bladder.