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Table of Contents
Year : 2019  |  Volume : 62  |  Issue : 5  |  Page : 188-195

Glycine tomentella hayata extract and its ingredient daidzin ameliorate cyclophosphamide-induced hemorrhagic cystitis and oxidative stress through the action of antioxidation, anti-fibrosis, and anti-inflammation

1 Department of Life Science, College of Science, National Taiwan Normal University, Taipei, Taiwan
2 Department of Life Science, College of Science, National Taiwan Normal University, Taipei; Department of Urology, Taipei Hospital, Ministry of Health and Welfare, New Taipei City, Taiwan
3 Department of Life Science, College of Science, National Taiwan Normal University, Taipei; Department of Urology, Cardinal Tien Hospital, New Taipei City, Taiwan

Date of Submission05-Sep-2019
Date of Decision20-Sep-2019
Date of Acceptance25-Sep-2019
Date of Web Publication24-Oct-2019

Correspondence Address:
Dr. Bing-Juin Chiang
Department of Urology, Cardinal Tien Hospital, New Taipei City 23148
Prof. Chiang-Ting Chien
School of Life Science, National Taiwan Normal University, No. 88, Tingzhou Road, Taipei City 11677
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/CJP.CJP_60_19

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We explored the therapeutic potential of intragastric administration of traditional Chinese medicine Glycine tomentella Hayata (I-Tiao-Gung [ITG]) extract and its major component Daidzin on cyclophosphamide (CYP)-induced cystitis, oxidative stress, fibrosis, inflammation, and bladder hyperactivity in rats. Female Wistar rats were divided into control, CYP (200 mg/kg), CYP+ITG (1.17 g/kg/day), and CYP+Daidzin (12.5 mg/kg/day) groups. We measured the voiding function by the transcystometrogram and evaluated the pathology with the hematoxylin and eosin and Masson stain. We determined the bladder reactive oxygen species (ROS) amount by an ultrasensitive chemiluminescence analyzer, the expression of 3-nitrotyrosine (3-NT) and NADPH oxidase 4 (NOX4) by Western blot and the expression of multiple cytokine profiles, including matrix metalloproteinase (MMP)-8 and tissue inhibitor of metalloproteinase (TIMP)-1 through a cytokine array. ITG extract contains 1.07% of Daidzin through high-performance liquid chromatography. The effect of ITG extract and Daidzin in scavenging hydrogen peroxide activity was more efficient than distilled water. CYP-induced higher urination frequency, shorter intercontraction interval, and lower maximal voiding pressure in the bladders and these symptoms were significantly ameliorated in CYP+ITG and CYP+Daidzin groups. The amount of in vivo bladder ROS and the expression of 3-NT and NOX4 expressions were significantly increased in CYP group but were efficiently decreased in the CYP+ITG and CYP+Daidzin groups. CYP-induced fibrosis, hemorrhage, leukocyte infiltration, and edema in the bladders were significantly attenuated in the CYP+ITG and CYP+Daidzin groups. These results suggested that ITG extract and its active component Daidzin effectively improved CYP-induced oxidative stress, inflammation, and fibrosis through inhibiting the MMP-8, TIMP-1, and oxidative stress.

Keywords: Cyclophosphamide, cystisis, daidzin, hyperactive bladder, I-Tiao-Gung, oxidative stress

How to cite this article:
Wu KC, Lin WY, Sung YT, Wu WY, Cheng YH, Chen TS, Chiang BJ, Chien CT. Glycine tomentella hayata extract and its ingredient daidzin ameliorate cyclophosphamide-induced hemorrhagic cystitis and oxidative stress through the action of antioxidation, anti-fibrosis, and anti-inflammation. Chin J Physiol 2019;62:188-95

How to cite this URL:
Wu KC, Lin WY, Sung YT, Wu WY, Cheng YH, Chen TS, Chiang BJ, Chien CT. Glycine tomentella hayata extract and its ingredient daidzin ameliorate cyclophosphamide-induced hemorrhagic cystitis and oxidative stress through the action of antioxidation, anti-fibrosis, and anti-inflammation. Chin J Physiol [serial online] 2019 [cited 2022 Dec 4];62:188-95. Available from: https://www.cjphysiology.org/text.asp?2019/62/5/188/269839

Kung-Chieh Wu and Wei-Yu Lin contributed equally to this work.

  Introduction Top

Lower urinary tract symptoms, including urine storage and voiding dysfunction, pain sensation, urgency, and postmicturition dribble, are a highly prevalent condition that influences the quality of life.[1],[2] Among these symptoms, urine storage dysfunction is believed to be a major problem.[3],[4] Overactive bladder (OAB), a widespread condition and easily ignored, is a symptom complex syndrome characterized by urine storage dysfunction. The syndrome includes symptoms such as urinary urgency, additional daytime frequency, and nocturia all with the absence of urinary tract infection, tumor, or urolithiasis.[3]

The factors lead to OAB are urethral outlet obstruction, bacterial infection, and urothelial damage. These factors are as well considered as the causes of urinary tract injury and chronic inflammation.[5] Bladder inflammation cause the production of reactive oxygen species (ROS), the main character in oxidative stress, and may eventually lead to bladder dysfunction,[6],[7] and bladder fibrosis. Matrix metalloproteinases (MMPs) are a family of enzymes involved in different processes such as modulation of inflammation, tissue remodeling and collagen processing.[8] The abnormal of MMPs and tissue inhibitor of metalloprotease (TIMPs) and/or dysregulation in MMPs/TIMPs are responsible for the process of fibrosis or proteolytic activity [8],[9],[10],[11],[12] and possibly contributes to the bladder diseases. MMPs establish a complex network, in which different enzymes may play opposite roles. In addition, specific family members may play different roles in different time points of the disease. For example, MMP-8, also known as collagenase-2, can digest native collagen, but its function in vivo seems to be more related to the control of the inflammatory response.[13] By the ability to cleave different cytokines and chemokines, MMP-8 promotes the initial onset and the later clearance of the neutrophilic inflammatory response and mice lacking MMP-8 show a delayed wound healing [14] and a decreased lung fibrosis.[15] Earlier evidence has indicated that cyclophosphamide (CYP) not only causes abnormal extracellular matrix (ECM) deposition, fibrosis but also leads to smooth muscle hyperreflexia in the bladder.[16] CYP was intraperitoneally injected to induce bladder hyperactivity in the rat model in other research.[17],[18] Therefore, we used CYP to induce bladder inflammation and fibrosis in a rat model of this study to explore the response of MMP-8 expression.

I-Tiao-Gung (ITG) (Glycine tomentella Hayata), a traditional Chinese herbal medicine, has been widely used in the treatment of rheumatic diseases and soreness in Taiwan.[19] ITG extract contains soy isoflavones, phytoestrogens, flavonoids, and phenolic substances that can diminish proinflammatory cytokines, scavenge free radicals, and inhibit lipid peroxidation.[19] Furthermore, soy isoflavones replacement has been reported to benefit detrusor overactivity in ovariectomized rats.[20] Our recent report has first demonstrated that ITG extract through its active component Daidzin effectively improved CYP-induced bladder contractile dysfunction by the action of restoring Phase 2 activity and inhibiting the expressions of purinergic P2 × 2, P2 × 3, and muscarinic M3 receptors.[21] However, to further explore the other protective mechanisms, we considered the antioxidant soy isoflavones-rich ITG may diminish ROS-evoked bladder oxidative stress, inflammation, and fibrosis leading to ameliorate CYP-induced hemorrhagic cystitis.

In this study, we aimed to determine the active components, therapeutic effects, and mechanisms of ITG extract on CYP-induced bladder inflammation, fibrosis, and oxidative injury.

  Materials and Methods Top


G. tomentella Hayata, also known as ITG, grew in the Xiguo mountain of Kinmen area in Taiwan and was provided by Kinmen County Agriculture Research Institute. We used the biennial dried main root of ITG in our study.

Preparation of I-Tiao-Gung extract

The method for extracting ITG was described previously.[19] In brief, the dry root of ITG was grounded, added ethanol, heated at 70°C for 2 h, and filtrated. After doing the previous step twice, the combined liquid extracts were lyophilized and obtained ITG extract. The ITG extract was then stored at 4°C until use.[19]

High-performance liquid chromatography analysis

The ITG extract was analyzed using high-performance liquid chromatography (HPLC, Hitachi system) consisting of a LaChrom Pump L-2130, a Programmable Autosampler L-2200, an Interface D-7000 and a LaChrom Diode Array Detector L-2455 (Merck, Vienna, Austria). The reagents of daidzin were purchased from Sigma Chemical Co., (St. Louis, MO., USA). Acetonitrile (HPLC grade) and DMSO (analytical reagent grade) were obtained from Merck (Darmstadt, Germany).


Twenty-four female Wistar rats (200–250 g body weight) purchased from BioLASCO (Taiwan Co. Ltd., Taipei, Taiwan) were housed with a consistent light cycle (light from 07:00–18:00). Food and water were provided ad libitum. All the surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Taiwan Normal University and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997), and Ethical Use Committee (Ethics number: 107033).

The rats were intraperitoneally anesthetized with urethane (1.2 g/kg body weight). Urethane was chosen for lacking ganglionic blocking properties.[21] The maintenance of deep anesthesia was determined by the persistence of miotic pupils as judged from frequent inspection.[21] Body temperature was maintained at 37°C with a heat lamp. After experiments, the anesthetized animals were sacrificed by intravenous injection of KCl.

Twenty-four rats were divided into control rats (Control, n = 6), CYP rats (CYP, n = 6), CYP treated with ITG rats (CYP+ITG, n = 6), and CYP treated with Daidzin rats (CYP+Daidzin, n = 6). The detailed protocols are shown in [Figure 1]a.
Figure 1: (a) Experimental protocol in four divided groups. Cyclophosphamide (200 mg) was intraperitoneally injected at the indicated time with a black arrow for 48 h. Treatment of I-Tiao-Gung extract or Daidzin was administered at time 0 and 24 h during 48 h of cyclophosphamide treatment. (b) High-performance liquid chromatography of Daidzin standard, (c) The original graph of high-performance liquid chromatography of the I-Tiao-Gung extract sample, (d) The antioxidant effect of I-Tiao-Gung extract in scavenging hydrogen peroxide amount, (e) The antioxidant effect of Daidzin in scavenging hydrogen peroxide amount. *P < 0.05 when compared to 0 mg/mL or Daidzin

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The dosage of I-Tiao-Gung and daidzin

Previous studies have indicated the safe dose of ITG extract was <2 g/kg/day in rats for 28 days.[22] Our preliminary data have evaluated the dosage at intragastrical 0.117 g/kg, 0.585 g/kg, 1.17 g/kg, and 3.34 g/kg on CYP-induced voiding dysfunction. We found ITG extract at 1.17 g/kg displayed the most efficient function on the improvement CYP-induced OAB. The intragastric dosage of 3.34 g/kg increased nonvoiding contractions in the bladders implicating a possible toxic effect. Therefore, our ITG extract contained 1.07% Daidzin (12.5 mg Daidzin in 1.17 g ITG), and we applied the ITG extract mixed with 1 ml distilled water (1.17 g/kg/day) to the CYP+ITG rats intragastrically. The Daidzin powder mixed with 1 mL of distilled water (12.5 mg/kg/day) was applied to the CYP+Daidzin rats, and the distilled water was given to the control rats.

In vitro and in vivo chemiluminescence recording for reactive oxygen species activity

The major ROS generated from activated leukocytes through the myeloperoxidase system is hydrogen peroxide (H2O2), which can initiate inflammation.[23] We evaluated the antioxidant H2O2 activity of ITG extract and Daidzin on the enhanced chemiluminescent (CL) signals from the H2O2-luminol mixture as described previously.[23]

The ROS produced in response to CYP stimulation was measured in the bladder in vivo by a CL method as previously described.[24] Briefly, the bladder of rats were intravenously administrated a superoxide anion probe, 2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo- [1,2-a] -pyrazin-3-one-hydrochloride (MCLA; 0.2 mg/ml/h, TCI-Ace, Tokyo Kogyo Co., Ltd., Tokyo, Japan) and detected with a Chemiluminescence Analyzing System (CLA-110, Tohoku Inc., Co., Sendai, Japan).

Cyclophosphamide-induced cystitis and bladder hyperactivity

CYP-induced hemorrhagic cystitis and bladder hyperactivity were intraperitoneally injected with CYP at a dose of 200 mg/kg, while the control group received saline.

Transcystometric model

We utilized a transcystometric technique to evaluate voiding response of CYP.[21] The following parameters of bladder responsiveness were measured: intercontraction interval (ICI, the time interval between two micturition cycles identified with active contractions [>10 mmHg]), maximal voiding pressure (MVP) and the micturition time spent from Phase 1 to Phase 4 contraction micturition time (MT).

Western blot

The expression levels of respective protein 3-nitrotyrosine (3-NT, #ab61392, Abcam's RabMb ® technology, Cambridge, UK) and NADPH oxidase 4 (NOX4, #MBS176126, Mybiosource, San Diego, USA) in the whole-bladder homogenates were analyzed using Western blot. Horseradish peroxidase conjugated goat anti-mouse IgG (#GGFC-90P, Immunology Consultants Laboratory, Inc., Portland, OR, USA) and goat anti-rabbit IgG (#406401, Biolegend, San Diego, CA, USA) were used as the secondary antibody. The density was determined semi-quantitatively by densitometry using an image analyzing system (Alpha Innotech, San Leandro, CA, USA).

Multiple cytokine antibody arrays

In response to toxicity, several inflammatory mediators such as cytokines and chemokines could be released by activated macrophages/Kupffer cells in the damaged bladder. Therefore, multiple cytokine expression levels were simultaneously detected and identified with the aid of RayBio ® rat cytokine protein array (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer's instructions.


Five micrometer sections of formalin-fixed bladders were stained with hematoxylin and eosin (H and E) for evaluating leukocytes infiltration, hemorrhage, and edema, Masson's trichrome for fibrosis.

Statistical analysis

All data were expressed as means ± standard error of the mean. Data were subjected to one-way analysis of variance, followed by Duncan's multiple range test for the assessment of the difference among groups. P < 0.05 was considered as statistically significant.

  Results Top

I-Tiao-Gung high-performance liquid chromatography analysis

Our results showed 1.07% of Daidzin in ITG extract [Figure 1]b and [Figure 1]c. Thus, Daidzin was selected as a major component and was applied to the animal experiment for evaluating CYP-induced bladder hyperactivity. ITG extract dose dependently and significantly reduced H2O2 amount [Figure 1]d as well as Daidzin in scavenging H2O2 amounts [Figure 1]e.

In vivo chemiluminescence recording for bladder reactive oxygen species parameters

The ROS amounts in CYP group significantly increased compared with the control group. CYP-induced bladder ROS decreased significantly in the CYP+ITG and CYP+Daidzin groups [Figure 2]a and [Figure 2]b.
Figure 2: Effect of I-Tiao-Gung extract or Daidzin on cyclophosphamide-induced bladder reactive oxygen species production in vivo of the rat urinary bladder and voiding parameters. The original data (a) and statistic data (b) of reactive oxygen species from the urinary bladder in the control, cyclophosphamide-treated, cyclophosphamide+I-Tiao-Gung, and cyclophosphamide+Daidzin groups are indicated. cyclophosphamide enhanced bladder reactive oxygen species and enhancement of reactive oxygen species was depressed by I-Tiao-Gung extract or Daidzin treatment. The urodynamic data in four groups of rats were indicated in c-e. (c) Intercontraction interval, (d) Maximal voiding pressure, and (e) Micturition time (the time from Phase 1 to Phase 4). *P < 0.05 when compared to Control group. #P < 0.05 when compared to cyclophosphamide group

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Daily ITG extract or Daidzin treatment in control rat did not display abnormal voiding parameters (nonpresented data). Our results showed the urinary frequency multiplied and the time of ICI shortened significantly in CYP group compared with the control group and the condition were cured with ITG extract or Daidzin treatment [Figure 2]c. MVP declined statistically in CYP, CYP+ITG, and Daidzin+CYP group compared with the control group [Figure 2]d. The time of MT augmented in CYP group was massively minimized with ITG extract or Daidzin treatment [Figure 2]e.

Western blot

3-NT expression was significantly enhanced in CYP group, whereas 3-NT expression was lower in the ITG extract or Daidzin-treated group [Figure 3]a. In addition, 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 3]b.
Figure 3: The expression of 3-NT and NOX4 proteins, in the control group, cyclophosphamide-induced groups, cyclophosphamide+I-Tiao-Gung group, cyclophosphamide+Daidzin group. The expression of 3-NT (a), NOX4 (b), in Control, cyclophosphamide, cyclophosphamide+I-Tiao-Gung, cyclophosphamide+Daidzin groups. All the statistic bars were obtained from n = 3 in each group. *P < 0.05 when compared to the Control group. #P < 0.05 when compared to cyclophosphamide group

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Cytokine array

CYP enhanced MMP-8 expression [Figure 4]a, whereas MMP-8 expression was lower in ITG extract or Daidzin-treated group. In addition, TIMP-1 generated higher in CYP group and was eliminated through ITG extract or Daidzin treatment [Figure 4]b.
Figure 4: (a) The typical expression of cytokine antibody array including matrix metalloproteinase-8, and tissue inhibitor of metalloprotease-1 proteins, in the control group, cyclophosphamide -induced groups, cyclophosphamide+I-Tiao-Gung group, cyclophosphamide+Daidzin group. (b) The statistical data of the expression of matrix metalloproteinase-8 and tissue inhibitor of metalloprotease-1 in Control, cyclophosphamide, cyclophosphamide+I-Tiao-Gung, cyclophosphamide+Daidzin groups. All the statistic bars were obtained from n = 3 in each group. *P < 0.05 when compared to Control group. #P < 0.05 when compared to cyclophosphamide group

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Bladder histology

Masson's trichrome stain [Figure 5]a showed severe fibrosis in CYP bladder and slight fibrosis in CYP+ITG and CYP+Daidzin group. Hemorrhage [Figure 5]b, leukocytes infiltration [Figure 5]c, and edema [Figure 5]d were found in the CYP bladders, whereas the condition was better in CYP+ITG, CYP+Daidzin bladders.
Figure 5: The effects of I-Tiao-Gung extract on the degree of fibrosis by Masson stain (a), hemorrhagic severity (b), leukocyte infiltration (c) and edema (d) of the rat urinary bladder in control group, cyclophosphamide-induced groups, cyclophosphamide+I-Tiao-Gung group, and cyclophosphamide+Daidzin group were demonstrated. A-1-A-4: Masson's trichrome staining. B-1-B-4: Hematoxylin and eosin staining of hemorrhage indicated with black arrows. C-1-C-4: Hematoxylin and eosin staining of leukocyte infiltration by blue stains. D-1-D-4: Hematoxylin and eosin staining of edema indicated with the black line, were indicated and were obtained from n = 6 in each group

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  Discussion Top

Recent studies demonstrated CYP leads to bladder hyperactivity. The increased voiding frequency was found 2 days after intraperitoneal injection with CYP [19],[25] and a significant reduction of voiding frequency was observed in the ITG and Daidzin-treated groups. Exaggerated ROS production may induce oxidative injury, inflammation, and voiding dysfunction in the bladders.[23],[24] NOX4 is a 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.[26],[27],[28] 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.[26] Our study also showed higher 3-NT expression in the CYP group, and lower 3-NT expression in the ITG and Daidzin-treated bladders. In addition, activation or high expression of NOX4 exaggerated ROS production.[27] CYP-treated bladder displayed the higher expression of NOX4 possibly and sequentially stimulating higher ROS formation in the CYP-treated urinary bladder.[28] 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.

Moreover, studies have shown ROS can activate MMPs, function as basement membrane and ECM degradation,[29] and can downregulate the endogenous inhibitors of MMPs (TIMPs).[30] 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.[31],[32] Zhang et al. demonstrated a upregulation of MMP-8 and/TIMP-1 expression in the left ventricles of rats with cardiac volume overload.[32] 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.[13] MMP-8 promotes the initial onset and the later clearance of the neutrophilic inflammatory response and mice lacking MMP-8 show a delayed wound healing [14] and a decreased lung fibrosis [15] MMP-8 is present in the initial stages of the acute inflammatory response and can, therefore, influence neutrophil recruitment.[33] The dysregulated enhancement of MMP-8 and TIMP-1 expression was found in myocardium with cardiac volume overload [32] and in our CYP bladders. Our present data were consistent with previous report that the elevation of MMP-8 and TIMP-1 contributed to inflammatory responses in the CYP group.[34] However, 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.[35],[36] These 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 through Masson's trichrome stain. However, significant recoveries were found in the bladders treated with ITG extract and Daidzin. In addition, studies have shown that the thickening of bladder wall may result from excessive production of profibrotic ECM and may provoke muscle growth, edema, and infiltration of immune cells and inflammatory substances.[37],[38],[39] We verified the edema and leukocytes infiltration in the bladders of CYP group, and the structures were ameliorated in the bladders of ITG and Daidzin-treated groups through H and E stain.

We speculated that ITG extract and daidzin may reduce the inflammation and the edema in the bladder through inhibiting the expression of lipoxygenase (LOX), cyclooxygenase-2 (COX-2), interleukin (IL)-1β, and IL-6 according to previous studies.[40] Some researches indicated that ITG extract inhibits inflammation through inhibiting 5-(5-LOX) and COX-2 pathway,[40] reducing inflammatory cytokines IL-1β, IL-6, and decreasing MMP-9 activity.[19] However, the anti-inflammatory mechanism and the change of edema through ITG is not fully understood and we will continuously investigate the impacts of ITG on inflammation and edema consequently.

  Conclusions Top

ITG extract can improve CYP-induced cystitis and bladder hyperactivity through the action of Daidzin to confront with oxidation, inflammation, and fibrosis and to downregulate MMP-8, TIMP-1, 3-NT, and NOX4 expression in CYP bladders.


This work was supported by the Ministry of Science and Technology of Republic of China (MOST107-2314-B-567-002, MOST107-2218-E-003-001) and research fund (201911) from Taipei Hospital.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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