The Experimental Study Of Particulate Matter On Bladder Function In A Rat Model By Aspiration Of Particles From The Pharynx And Its Mechanism

Objective:Establish of a rat model by aspiration of particles from the pharynx, to determine whetherdiesel exhaust particles (DEPs) could be a toxic agent to the bladder.Methods:Rats were randomly assigned into two time groups of forty each group, one month andthree-month exposure group. Then each group was randomly divided into five groups ofdifferent concentrations namely the control group, low dose, medium dose, high dose,and super-high dose exposure groups (group A-E), eight rats respectively. The fourexposure groups were respectively given a volume of30μl SRM1649a suspension at0.28(B),1.58(C),5.61(D),21.03(E) μg/μl，the control group was inoculated with30μlPBS on pharynx posterior wall by sample pipettor beginning at day one when rats wereacclimatized for one week, then repeated per triduum for a total of10or30times in onemonth or three-month exposure group. Three days after the last exposure, rats weredeprived of food for24h and then prepared for experimental procedure. When the ratswere sacrificed, morphologic changes of the urothelium were investigated. Theantioxidase activity and the levels of lipid peroxidation in the bladder were assayed; Theexpression of Cx26、Cx32、Cx36、Cx37、Cx40、Cx43and Cx45mRNA in bladder weredetected by RT-PCR techniques, in transcript levels; The levels of Cx43protein inbladder were detected by Western blotting and Q-PCR; Primary cell cultures were set upfrom fragments of rat bladder at super-high dose in three-month group. After cultureddetrusor cells were loaded with the fluorescent dye6-CFDA, the function of GJIC in thecultured bladder detrusor cells were detected by fluorescence redistribution afterphotobleaching (FRAP) techniques; The effect of18β-GA at various concentrations on cultured rat bladder detrusor cells of super-high dose in three-month group were observedby FRAP techniques under laser scanning confocal microscope (LSCM). Meanwhile, theeffect of18β-GA of the same concentrations at various times on cultured rat bladderdetrusor cells of super-high dose in three-month group were also observed by FRAPtechniques in this study.Results:1. Rats exposure model established by aspiration of particles from the pharynx is simple,easy to operate and good repeatability.2. In the three-month group, DEPs at doses of21.03μg/μl insulted the structuralintegrity of surface glycosaminoglycans, widened the gap between urothelial cells,increased levels of lipid peroxidation, and decreased antioxidase activities in theurinary bladder (p<0.05). Furthermore, DEPs at doses of5.61μg/μl decreasedglutathione, catalase, and glutathione peroxidase activities (p<0.05).3. RT-PCR analysis of control and exposure group rat bladder RNA demonstrated thepresence of a transcript for six connexins (Cx26, Cx32, Cx37, Cx40, Cx43, and Cx45but not for Cx36), the levels of Cx43, Cx40mRNA in doses of21.03μg/μl ofthree-month group was higher than in the control groups, increased2.9and3.2timesrespectively (p<0.05). But, the rest Cx mRNA expression were no significantdifferences (p<0.05). The level of Cx43protein expression in detrusor tissue of thesuper-high dose in three-month group was notably higher than in the control group,increased2.4times (p<0.05).4. By collagenase digestion of rat bladder detrusor cells in primary culture, the cycle isshort, and high purity detrusor cells can be obtained. Cultured detrusor cells loadedwith6-CFDA fluorescence probes grew to a flattened simple form. The functions ofGJIC in the cultured rat bladder detrusor cells of the super-high dose in three-monthgroup and control groups were detected by FRAP techniques. It shows that thefluorescence intensity was gradually recovered at different times after bleaching inthe super-high dose in three-month group. The mean fluorescence recovery rates in doses of21.03μg/μl of three-month group (4minutes) were (36.4±0.73)%. In contrastto the super-high dose in three-month group, the fluorescence recovery of thebleached cells in the control groups were not obvious in the same time. The meanfluorescence recovery rates of the control groups (4minutes) were (11.4±0.94)%.5. The effect of18β-GA at various concentrations on cultured rat bladder detrusor cellsof super-high dose in three-month group were observed by FRAP techniques. Theseresults showed that no time-dependent in the functional change of GJIC at super-highdose in three-month group cells.Conclusion:1. These results led to the conclusion that DEPs were a toxic agent in the bladder. Thetoxic effects might be attributed to oxidative damage mediated by pro-oxidant/antioxidant imbalance or excessive free radicals.2. Exposed to high concentrations of DEPs, bladder tissue may regulate the Cx gene andprotein expression to adapt to environmental changes. The mechanism may be haverelationship with DEPs lead to bladder epithelial cell gap widened and imblance ofthe environmental redox system in the bladder tissue, but further studies are needed toconfirm.3. The gap junction is an important structural basis for detrusor excitation transmissionbetween adjacent cells. It has demonstrated from a functional perspective thatdetrusor GJIC enhancement in dose of21.03μg/μl of three-month group by FRAPtechnique. One of the pathological mechanisms leading to bladder toxic effects byDEPs may be is GJIC enhancements between detrusor cells.4. At cell levels, the GJIC of super-high dose in three-month group was inhibited by18β-glycyrrhetinic acid (18β-GA), as a gap junction blocker. These results maycontribute to new concepts in the treatment of bladder disease caused by DEPs in thefuture.