3-1 ITG-HPLC analysis
Our results showed 1.07% of Daidzin and 0.77% of Daidzein (Figures 1B-1D). Thus, Daidzin was selected as a major component and was applied in the animal experiment for evaluating CYP-induced bladder hyperactivity. ITG dose-dependently and significantly reduced H2O2 amount (Figure 1E). Daidzin inhibited H2O2 amounts. Daidzin is more efficient than Daidzein in scavenging H2O2 amount (Figure 1F).
3-2 The effect of ITG on voiding parameters
Daily ITG or Daidzin treatment in control rat did not display abnormal voiding parameters (unpresented data). Our results showed that the level of PTH in CYP group was significantly decreased as compared to the control group. The decreased PTH by CYP was significantly recovered by ITG treatment (Figure 2B). A significant decrease of ICI and a significant increase of urinary frequency were found in CYP group vs. control group (Figures 2C and 2D). The
improvement of bladder hyperactivity was noted in the CYP+ITG, CYP+Daidzin, ITG+CYP groups (Figures 2C and 2D) and CYP+Daidzein group (Supplemental figure). The BP in CYP, CYP+ITG and CYP+Daidzin group was significantly reduced vs. control group (Figure 2E). MT (time spent from Phase 1 to Phase 4 contraction as described in Figure 3F) in CYP group was significantly increased vs.
control group (Figure 2F). The CYP-increased MT was significantly depressed by ITG or Daidzin treatment (Figure 2F). MVP in CYP, CYP+ITG and ITG+CYP was significantly decreased vs. control group (Figure 2G). AM in CYP group was significantly reduced vs. control group (Figure 2H). The significant improvement of AM was found in the CYP+Daidzin group vs. CYP group (Figure 2H).
3-3 The effect of ITG and bladder hyperactivity
The voiding frequency in CYP group (Figure 3B) was significantly increased vs. control group (Figure 3A). CYP-enhanced bladder
hyperactivity was significantly depressed in the CYP+ITG (Figure 3C), CYP+Daidzin (Figure 3D) and ITG+CYP (Figure 3E) group.
3-4 The effect of ITG on voiding cycle and EMG activity
An increased voiding frequency associated with a depressed EUS-EMG response were found in CYP rats (Figure 3G) vs. control rats (Figure 3F). These responses were partly recovered in CYP+ITG (Figure 3H), CYP+Daidzin (Figure 3I) and ITG+CYP (Figure 3J) rats. A normal voiding cycle includes four voiding contractile phases (Figure 3F). CYP efficiently depressed the response of Phase 2 duration, peak EUS-EMG activity and silent/active component ratio (Figures 3K-3M). These depressed responses were partly recovered by ITG or Daidzin treatment.
3-5 The effect of ITG on purinergic receptors
The expression of P2X2 (Figure 4A) and P2X3 (Figure 4B) was significantly upregulated in CYP bladders. The upregulation of P2X2 and P2X3 by CYP was significantly depressed by ITG or Daidzin treatment.
3-6 The effect of ITG on muscarinic receptors
The M2 expression was similar among these five groups (Figure 4C).
CYP significantly enhanced M3 expression, whereas the CYP-enhanced M3 expression was significantly depressed by ITG or Daidzin treatment (Figure 4D).
3-7 The effect of ITG and bladder inflammatory and fibrosis and MDA
The increased hemorrhage (Figure 5A), edema (Figure 5B) and leukocytes infiltration (Figure 5C) were found in the CYP bladders.
CYP+ITG, CYP+Daidzin, and ITG+CYP bladders significantly decreased hemorrhage (Figure 5E), edema (Figure 5F) and leukocytes infiltration (Figure 5G). Masson’s trichrome staining showed increased fibrosis in CYP bladder and decreased fibrosis in CYP+ITG, CYP+Daidzin, and ITG+CYP bladders (Figure 5D). CYP significantly increased bladder MDA level, whereas the increased MDA level wase significantly reduced by ITG or Daidzin treatment (Figure 5H).
3-8 The effect of ITG and purinergic and muscarinic receptors and mast cell and hematuria
M2 expression was similar among these five groups (Figures 6A and 6G). CYP enhanced M3 expression, whereas CYP-enhanced M3 expression was decreased by ITG or Daidzin treatment (Figures 6B and 6H). CYP significantly enhanced the expression of P2X2 (Figures 6C and 6I) and P2X3 (Figures 6D and 6J), whereas the enhanced expression of P2X2 and P2X3 was significantly inhibited by ITG or Daidzin treatment (Figures 6I and 6J). Toluidine blue staining showed increased mast cells in CYP group vs. control group (Figure 6E). The mast cells number wase significantly reduced in CYP+ITG, CYP+Daidzin, and ITG+CYP groups vs. CYP group (Figure 6K). The appearance of hematuria was found in the CYP group and the response was inhibited in CYP+ITG, CYP+Daidzin, and ITG+CYP groups (Figure 5J).
3-9
In vivo chemiluminescence recording for bladder ROSparameters
The ROS amounts in CYP group was significantly increased compared with the control group. CYP-induced bladder ROS was decreased significantly in the CYP+ITG and CYP+Daidzin groups (Figures 7 A and B).
3-10 The effect of ITG on 3-NT and NOX4
3-NT expression was significantly enhanced in CYP group, whereas 3-NT expression was lower in the ITG extract or Daidzin treated group (Figure 8A). Additionally, the NOX4 expression was significantly elevated in CYP group as compared to control group and was enormously depressed by ITG extract or Daidzin treatment in CYP+ITG and Daidzin+CYP groups (Figure 8B).
3-11 Cytokine array
MMP-8 expression was significantly enhanced in the CYP group as compared to control group (Figure 9A), whereas the enhanced MMP-8 expression was significantly reduced in ITG extract or Daidzin treated group. Additionally, TIMP-1 expression was significantly higher in CYP group and was significantly depressed in the ITG+CYP group or Daidzin+CYP group (Figure 9B).
Chapter 4. Discussion and Conclusion
4-1 The treatment of ITG and Daidzin on CYP-induced bladder hyperactivity
CYP significantly increased bladder hyperactivity. The increased voiding frequency was found 2 days after intraperitoneal injection with CYP28,36 and a significant reduction in voiding frequency was observed by ITG or Daidzin treatment. Dietary Daidzin can efficiently improve detrusor overactivity of ovariectomized rats.2In our prepared ITG extract, Daidzein is also contained in ITG extract at approximately 70% level of Daidzin. Our in vitro data showed that the capability of anti-oxidant activity in Daidzin was more efficient than in Daidzein (Figure 1F). Our in vivo data showed that the recovery of the shorten ICI level was
significantly higher in Daidzin than in Daidzein (70% value of Daidzin in Supplemental figure) implicating Daidzin is more efficient than Daidzein in improvement of CYP-induced bladder hyperactivity. We found that Daidzin alone is more effective than ITG extract (mixture of Daidzin and Daidzein) in improving CYP-induced bladderhyperactivity possibly due
to some components affecting purinergic and cholinergic neurotransmission, proinflammatory or pro-oxidant signaling existing in the ITG extract. Therefore, based on these data, we suggest that Daidzin may play a more important role than Daidzein in ITG extract to ameloriate CYP-induced bladder dysfunction. In addition, ITG+CYP also demonstrated similar improvement with CYP+ITG. We suggest that the use of ITG extract can ameliorate CYP-induced bladder dysfunction through the preconditioning or conditioning route in future.
4-2 The effect of ITG and Daidzin on CYP-induced voiding dysfunction and EUS-EMG activity
The efficient voiding function depends on the co-ordinated activity of smooth muscles (urinary bladder and urethra) and striated muscles (EUS) in the lower urinary tract and can be evaluated by the techniques of transcystometrogram and EUS-EMG recording.13,37A micturition cycle can be regulated by purinergic receptors-mediated Phase 1 and muscarinic receptors-mediated Phase 2 contraction.13 The increased IVP
in Phase 1 is associated with increased tonic pelvic efferent nervous activity and tonic EUS-EMG activity. Phase 2 characterized by the bursting of pelvic efferent nervous activity and EUS-EMG activity is responsible for the bladder emptying. Our data delineated CYP-induced voiding dysfunction is ascribed to the bursting EUS activity being converted to tonic EUS activity and the depressed silent/active component ratio possibly by the CYP toxicity to the pelvic and pudenal nerves and muscles. Increase in active period of EUS-EMG is likely to increase MT in CYP group compared to control group. ITG and its active component, Daidzin, can efficiently restore Phase 2 contraction, EUS-EMG activity and silent/active component ratio to ameliorate CYP-induced bladder dysfunction.
4-3 The effect of CYP-induced bladder hyperactivity in purinergic receptors and protein expression
ATP plays a role in sensory neurotrnamission of purinergic receptor, which in turn causes voiding reflexes.12 The exaggerated release of ATP
in the urothelial epithelium of neurogenic bladder leads to the activation of P2X2 and P2X3 receptors resulting in further neuronal activation and bladder hyperactivity.37,38 Increased P2X3 expression is also found in several types of neurogenic hyperactive bladder.11,39,40 The expressions of P2X2 and P2X3 receptors were significantly increased in our CYP bladders. The expressions of P2X3 and P2X2/3 receptors were increased significantly in the neurogenic bladders.41,42 Additionally, upregulation of P2X2 receptor promoted detrusor hyperactivity.43ITG extract or Daidzin efficiently and completely inhibited the upregulation of P2X2 and P2X3.
We suggest the application of ITG and Daidzin can reduce the expression of P2X2 and P2X3 subsequently improving bladder hyperactivity.
4-4 The effect of CYP-induced bladder hyperactivity in muscarinic receptors and protein expression
The expectable treatment is to suppress the exaggerated contractility of detrusor and to inhibit bladder hyperactivity.11,44 Mirabegron, a β3-adrenoceptor agonist, has been tested in the clinical trials for the
treatment of overactive bladder for its action to relax detrusor.45 M2 seems to play no role in CYP induced bladder hyperactivity because M2 expression is similar among these five groups. Overexpression of M3 receptors in detrusor contributes to bladder hyperactivity.34 Our data showed CYP enhanced M3 receptor expression and the enhanced M3 expression was significantly and partially depressed by ITG or Daidzin.
4-5 Effect of CYP-induced bladder inflammation triggered 3-NT or NOX4 expression and ROS accumulation
Exaggerated ROS production may induce oxidative injury, inflammation and voiding dysfunction in the bladders.33,46 NOX4 is an pro-oxidant enzyme for triggering ROS production and 3-NT, a product of tyrosine nitration mediated by ROS, is identified as an indicator or marker of cell damage, oxidative stress and inflammation.47-49 Previous studies have shown that CYP induced bladder inflammation triggered 3-NT and ROS accumulation in the urinary bladder, and further led to bladder hyperactivity.47Our study also showed higher 3-NT expression in
the CYP group and a lower 3-NT expression in the ITG and Daidzin treated bladders. In addition, activation or high expression of NOX4 exaggerated ROS production.48 CYP-treated bladder displayed the higher expression of NOX4 possibly and sesquentially stimulating higher ROS formation in the CYP-treated urinary bladder.49 Our data indicated CYP elevated NOX4 and 3-NT expression that was significantly attenuated through ITG extract or Daidzin treatment in the damaged bladders implicating the therapeutic potential of ITG in reducing oxidative injury.
4-6 Effect of CYP-induced bladder inflammation triggered MMP-8 or TIMP-1 expression
Moreover, studies have shown ROS can activate MMPs, function as basement membrane and extracellular matrix degradation,50 and can downregulate the endogenous inhibitors of MMPs (TIMPs).51 One previous report stated that some MMPs are anti-fibrotic, while other MMPs can have pro-fibrotic functions. Some studies have reported that CYP is related to MMPs and TIMPs expression.52,53 Zhang et al. (2013)
demonstrated a upregulation of MMP-8 and/TIMP-1 expression in the left ventricles of rats with cardiac volume overload.53 It is suggested that specific family members of MMPs may play different roles in different time points of the disease and in in vitro or in vivo condition. MMP-8 can trigger proteolytic activity, but its function in vivo seems to be more related to the control of the inflammatory response.24 MMP-8 promotes the initial onset and the later clearance of the neutrophilic inflammatory response and mice lacking MMP-8 show a delayed wound healing25and a decreased lung fibrosis26 MMP-8 is present in the initial stages of the acute inflammatory response and can therefore influence neutrophil recruitment.54 The dysregulated enhancement of MMP-8 and TIMP-1 expression was found in myocardium with cardiac volume overload53and in our CYP bladders. Our present data was consistent with previous report that the elevation of MMP-8 and TIMP-1 contributed to inflammatory responses in the CYP group.55However, the expression of MMP-8 and TIMP-1 was rarely observed in the ITG extract and Daidzin groups that indicated ITG extract and Daidzin could efficiently inhibit
MMP-8 and TIMP-1 expression, reduce inflammation and subsequently improve bladder hyperactivity.
The higher expression of TIMP-1 resulted in the inhibition of MMP activity and the accumulation of matrix proteins in extracellular space.56,57These studies concluded CYP may lead to bladder fibrosis, the production of MMPs and TIMP in bladder tissue and the deposition of collagen in ECM. We confirmed CYP induced bladder fibrosis via Masson’s trichrome stain. However, significant recoveries were found in the bladders treated with ITG extract and Daidzin. Additionally, studies have shown that the thickening of bladder wall may result from excessive production of pro-fibrotic ECM and may provoke muscle growth, edema, and infiltration of immune cells and inflammatory substances.58-60
4-7 Conclusions
ITG can improve CYP-induced cystitis and bladder hyperactivity through the action of Daidzin to confer ant-oxidation, anti-inflammation, restore Phase 2 activity by maintaining the balance between detrusor and
urethral and downregulate P2X2, P2X3, M3, MMP-8, TIMP-1, 3-NT and NOX4 expression in CYP bladders (Figure 10).
4-8 Future Works
ITG can inhibit inflammation through 5-lipoxygenase (5-LOX) and Cyclooxygenase-2 (COX-2) pathway61 and reduce inflammatory cytokines IL-1β, and IL-6 release.1 Our data presented the increased mast cells, leukocytes infiltration, hemorrhage and edema in the CYP bladders, but these inflammatory responses were all depressed by ITG or Daidzin treatment. We speculated that the inflammation can be reduced via the inhibition of LOX-COX-2-IL-1β-IL-6 pathway. However, the detailed mechanism for the reduction of leukocytes and mast cells are required determined further.
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