Scutellaria baicalensis Alleviates Cantharidin-Induced Rat Hemorrhagic Cystitis through Inhibition of Cyclooxygenase-2 Overexpression

Cantharidin, an active component in mylabris, is used in traditional Chinese medicine (TCM) to treat scabies and hepatoma, but accompanied by hemorrhagic cystitis. Evidence shows that cantharidin induces human bladder carcinoma cell death through COX-2 overexpression in vitro. In TCM, Scutellaria baicalensis is usually used to cure mylabris-induced hematuria. This work was undertaken to determine the mechanisms of cantharidin-induced rat hemorrhagic cystitis and explore the uroprotective effect of S. baicalensis. In vitro results showed cantharidin could induce cytotoxicity through prostaglandin (PG)E2 overproduction of T24 cells. Boiling-water extract of S. baicalensis (SB-WE) could significantly inhibit PGE2 production and COX-2 expression in lipo-polysaccharide-induced RAW 264.7 cells, indicating obvious anti-inflammatory abilities. In vivo results indicated that cantharidin caused rat hemorrhagic cystitis with hematuria via c-Fos and COX-2 overexpression. SB-WE was given orally to cantharidin-treated rats, whereby hematuria level, elevated PGE2 and COX-2 protein overexpression were significantly and dose-dependently inhibited by SB-WE. The anti-inflammatory components of SB-WE are baicalin and wogonin, whose contents were 200.95 ± 2.00 and 31.93 ± 0.26 μg/mg, respectively. In conclusion, cantharidin induces rat cystitis through c-Fos and COX-2 over-expression and S. baicalensis can prevent the resulting hematuria because of its anti-inflammatory effects.


Introduction
Cantharidin is a major component of mylabris (Mylabris phalerata Pallas), commonly known as Spanish Fly. In Europe and India, mylabris is used as an aphrodisiac. In traditional Chinese medicine (TCM), people use it to treat scabies and hepatoma [1,2]. However, people who take an overdose of mylabris will be poisoned by cantharidin. According to clinical case reports, cantharidin poisoning results in burning of the mouth, hematemesis, hepatomegaly, irritation of the genitourinary tract, hematuria, and dysuria [1,3,4]. Moreover, in a previous study we found that cantharidin induced secondary necrosis and cyclooxygenase (COX)-2 overexpression in bladder epithelial cells [5].
Nowadays, few study discuss rat hemorrhagic cystitis. Researchers usually use cyclophosphamide and ifosfamide, which are DNA-alkylating agents used in cancer therapy, as animal cystitis inducers. Metabolites of cyclophosphamide and ifosfamide can induce urinary bladder irritation and hematuria through COX-2 and inducible nitric oxide synthase (iNOS) overexpression [19,20]. Based on the above reasons, we assumed that cantharidin might cause bladder inflammation and then induce hematuria, via a mechanism which possibly the same as that for cyclophosphamide and ifosfamide.
In this study, we tried to establish a cantharidin-induced rat hematuria model by directly injecting cantharidin into the bladder of female Wistar rats and discuss the mechanism of its irritation. The boiling-water extract of S. baicalensis (SB-WE) was also used to treat cantharidin-induced rat hematuria and we explored its uroprotective mechanism. In vitro nitric oxide (NO) and prostaglandin (PG)E 2 inhibition abilities and in vivo immunohistochemistry, hematuria level and COX-2 expression of rat bladder were used to evaluate the uroprotection of SB-WE. Besides, the quality of the SB-WE was monitored by the levels of two substance markers, baicalin and wogonin, with a high-performance liquid chromatographic (HPLC) method.

Cantharidin Induced Rat Hemorrhagic Cystitis Through Initiating Urinary Bladder Inflammation
In an in vivo assay, cantharidin (0.5 mg/kg) was directly injected into the bladder of female rats, where it induced severe hematuria and dysuria after 16 h ( Figure 3). The pathology examination showed that cantharidin induced colliquative necrosis of the urinary bladder epithelium and induction of neutrophil infiltration in cantharidin-treated Wistar rats ( Figure 4A). Besides, the immunohistochemistry results showed that c-Fos, an inflammation-related transcription factor, was significantly increased in rat bladder after cantharidin injection ( Figure 4B). The urinary bladders were homogenized and protein extracted for the Western blot analysis.  Figure 5 shows that the COX-2 expression level in the cantharidin-treated group was 1.5 fold higher than in the blank group.

Anti-Inflammatory Effects of Boiling-Water Extracts of S. baicalensis (SB-WE)
To investigate the anti-inflammatory effects of SB-WE, a LPS-induced RAW 264.7 cell model was used. Results showed that NO and PGE 2 production by LPS-induced RAW 264.7 cells was significantly inhibited by the SB-WE in a dose-dependent manner, and the respective 50% inhibitory concentration (IC 50 ) values were 79.86 and 35.02 μg/mL ( Figure 6).

Uroprotective Effects of Boiling-Water Extracts of S. baicalensis (SB-WE)
As shown in Figure 3, the SB-WE dose-dependently alleviated the hematuria and increased the urine volume, indicating that the toxicity of cantharidin was reduced by S. baicalensis. The hematuria level was reduced from 3+ to 1+ in cantharidin-treated group after oral administration of SB-WE (200 mg/kg). Urine volume in the SB-WE group (200 mg/kg) was about 1.5 fold higher than the cantharidin-treated group (4.0 mL in cantharidin-treated group and 7.5 mL in SB-WE group). In histological analysis, the colliquative necrosis of the urinary bladder epithelium and neutrophil infiltration were attenuated in a dose-dependent manner by administration of SB-WE ( Figure 4A).
Besides, the signal of c-Fos was also reduced by the SB-WE ( Figure 4B, solid black triangles). As shown in Figure 5A, the cantharidin-induced COX-2 overexpression was alleviated by treatment with different dosages of the SB-WE ( Figure 5). The above results indicated that cantharidin-induced hematuria and dysuria might be correlated with an inflammatory pathway. The SB-WE displayed significant anti-inflammatory abilities and contributed to prevent cantharidin-induced hematuria in Wistar rats.

Quality Control of the Boiling-Water Extract of Scutellaria baicalensis (SB-WE)
The HPLC chromatogram profile of the SB-WE is shown in Figure 7. The respective retention times of baicalin and wogonin were 16.5 and 40.9 min. The respective contents of baicalin and wogonin in the SB-WE were 200.95 ± 2.00 and 31.93 ± 0.26 μg/mg.

Discussion
Cantharidin is a sesquiterpenoid toxicant and can be extracted from many species of blister beetle. In China, mylabris (Mylabris phalerata Pallas) is a kind of blister beetle used for anti-cancer or anti-arthritic drugs [21]. However, the therapeutic threshold of mylabris is very narrow. Many adverse reactions of mylabris were reported in clinical use [22][23][24]. According to case reports of cantharidin poisoning, cantharidin induces fever and hematuria in intoxicated patients [25]. However, the mechanisms of cantharidin-induced fever and hematuria are not clear. As shown in Figure 2, cantharidin induced PGE 2 production and necrosis in T24 cells which were alleviated by NS398, a selective inhibitor of COX-2. Evidence showed that COX-2-dependent cell death was also found in apoptosis-resistant cancer cells treated with an endogenous cannabinoid and anandamide [26]. Hence, we suggest that the possible mechanism of fever and hematuria induced by cantharidin is through induction of COX-2 overexpression by bladder epithelial cells.
In the discovery of uroprotective agents, cyclophosphamide and ifosfamide, two well-known DNA-alkylating agents, are commonly used as hematuria inducers. Metabolites of these two anti-cancer agents, acrolein and 4-hydroxyifosfamide, had been confirmed to be a potential inducers of urothelial irritation. Evidence has shown that hemorrhagic cystitis occurred by directly damaging with these two metabolized products, while lead to the ulceration, edema, neovascularization and necrosis of the bladder [27,28]. Evidence indicates that it takes 12-24 h to induce hemorrhagic cystitis after treatment with cyclophosphamide and ifosfamide [19,20]. In addition, cantharidin could rapidly induce blisters on the skin with topical exposure [29]. Cantharidin is also used as an inflammatory inducer to establish a new animal inflammation model [30], but cantharidin has a high systemic toxicity and causes diarrhea, congestion of the gastrointestinal tract and kidneys in vivo [31,32]. In this study, cantharidin was used to induce bladder inflammation and hematuria via direct injection into rat bladder without causing any systemic toxicity within only one day (Figures 3 and 4). After that, the uroprotective effects of S. baicalensis were evaluated on the basis of this acute rat hemorrhagic cystitis model. Firstly, results showed that SB-WE inhibited NO and PGE 2 production by LPS-induced RAW 264.7 cells, indicating that SB-WE displayed strong anti-inflammatory abilities in vitro ( Figure 6). Secondly, SB-WE also could reduce cantharidin-induced inflammation and hematuria through inhibition of c-Fos and COX-2 expressions in an in vivo assay. In TCM, S. baicalensis is a medicine widely used as an anti-inflammatory therapy and the characteristic of S. baicalensis is to clear internal heat. In addition, S. baicalensis is widely used as an anti-pyretic and detoxifying herb and is reported to have anti-cancer, anti-oxidative, and anti-inflammatory effects [14,17,21,33]. A study by Zhang et al. showed that S. baicalensis could inhibit tumor cell proliferation via suppression of COX-2 expression [21]. Our results show that the obvious COX-2 inhibition abilities of S. baicalensis contributed to the uroprotective effects against cantharidin-induced hemorrhagic cystitis. Baicalin and wogonin, the principal compounds in root of S. baicalensis, display significantly anti-inflammatory effects and have been used as the quality control indicators of the boiling-water extract.
Taken together, we first established a pharmacological animal model for discovering uroprotective agents. The mechanism of cantharidin-induced rat hemorrhagic cystitis was through overexpression of inflammatory-related mediators, c-Fos and COX-2. S. baicalensis, a traditional anti-inflammatory herb, displayed significant uroprotective effects against cantharidin-induced hematuria via inhibition of c-Fos and COX-2 overexpression.

Animals
Female Wistar rats (200~250 g) were bought from BioLASCO Taiwan (Yilan County, Taiwan), and maintained at 21 ± 2 °C with food and water ad libitum, and kept on a 12-h light/12-h dark cycle. All Wistar rats used in this experiment were cared for according to the Ethical Regulations on Animal Research of Taipei Medical University (approval No.: LAC-98-0050).

Isolation of Cantharidin
Dried mylabris (Chinese blister beetles) was bought from a traditional Chinese medicine store in Taipei, and voucher specimens (MP-002) were deposited in the Graduate Institute of Pharmacognosy Science, Taipei Medical University. The isolation procedure was the same as in our previous study [5]. The structure of cantharidin is shown in Figure 1.

Preparation and Quality Control of the Boiling Water Extract of S. baicalensis (SB-WE)
Dried roots of S. baicalensis were purchased from a traditional Chinese medicine store in Taipei and identified by Dr. Hsien-Chang Chang. Voucher specimens (SB-001) were deposited in the Graduate Institute of Pharmacognosy Science, Taipei Medical University. The preparation method was modified from our previous study [34]. Radices of S. baicalensis were immersed in purified deionized water and boiled for at least 30 min until half of the original amount was left. Aqueous solutions were then filtered, and freeze-dried under vacuum.
Two substance markers, baicalin and wogonin, were used to evaluate the quality of the boiling water extract of S. baicalensis (SB-WE). The HPLC equipment comprised an SCL-10Avp System Controller, an SPD-M10A Diode Array Detector, an LC-10ATvp Liquid Chromatograph Pump, an SIL-10Avp Auto Injector, FCV-10Avp Flow-Channel Selection Valves (Shimadzu, Tokyo, Japan), and an ERC-3415 Degasser (ERC, Altegolfsheim, Regensburg, Germany). The stationary phase consisted of a Purospher ® STAR RP-18e reversed-phase column (5 μm, 4 mm i.d. × 250 mm, Merck). Acetonitrile-water was used as the mobile phase in the gradient mode as follows: Acetonitrile: 0~17 min, 40%, 17~45 min, 40% to 100%. The flow rate was 1 mL/min, and the oven temperature was maintained at 40 °C. We used a UV wavelength of 280 nm to detect the baicalin and wogonin.

Cytotoxicity, PGE 2 and NO Inhibitory Assay
The protocols of the cytotoxicity, PGE 2 , and NO inhibitory assays were modified from our previous study [35]. RAW 2624.7 cells (4 × 10 5 cells/mL) were seeded on 96-well plates and co-treated with LPS (500 ng/mL) and the SB-WE. T24 cells (1 × 10 5 cells/mL) were seeded on 96-well plates and co-treated with cantharidin and NS398. After 18 h incubation, cytotoxicity was examined by an MTT assay, and the cell culture supernatant was collected for PGE 2 detection using a commercial assay kit (Assay Designs, Farmingdale, NY, USA). NO was measured as nitrite production in the medium after 24 h of incubation with or without the SB-WE, Briefly, nitrate in the medium was converted to nitrite and measured spectrophotometrically at 540 nm after the Griess reaction.

Cantharidin Induced Hemorrhagic Cystitis in Wistar Rats
Wistar rats were starved for 24 h with free access to water and anesthetized with Zoletil ® (Virbac, Carros cedex, France). The urinary bladders of the rats were instilled with cantharidin (0.5 mg/kg) via a transurethral injection with a 4-Fr. ureteral catheter (BARD, Murray Hill, NJ, USA). After 1 h, cantharidin-treated rats were orally administrated SB-WE (50, 100, 200, and 400 mg/kg). Each group had five test rats. Urine was continuously collected for 16 h after administration of the SB-WE, and then the rats were sacrificed to obtain the urinary bladders. Hematuria levels were analyzed by a PocketChem TM UA analyzer (Arkray, Tokyo, Japan). According to the operation manual, hematuria levels could be divided into four levels: 0+, 1+, 2+ and 3+ depending on the hemoglobulin content in urine. Urinary bladders were fixed in 10% paraformaldehyde, gradually dehydrated in ethanol, and embedded in paraffin. Immunohistochemical staining for c-Fos was performed on formalin-fixed, paraffin-embedded tissue sections. Staining was done using conditions recommended by the vendor. The protocol of COX-2 protein expression was modified from our previous study [5]. COX-2 Protein expression levels were analyzed with the AlphaImager Imaging System.

Statistical Analysis
Data are presented as the mean and standard deviation (SD) and were analyzed with Student's t-test and One-Way ANOVA by SPSS software. The in vitro and in vivo assays were performed by 3 and 5 independent trials, respectively. The significance level used for statistic analysis displayed as: * p < 0.05, ** p < 0.001.

Conclusions
In summary, we found that cantharidin-induced rat hemorrhagic cystitis through up-regulating expression of COX-2 and the inflammation-related transcription factor, c-Fos. In addition, S. baicalensis extracts could reduce the cantharidin-induced cytotoxicity, COX-2, PGE 2 and c-Fos expression in vitro and in vivo. Our results could be used as a drug screening platform to discover novel uroprotective drugs.