Evaluation of Chinese medicine Qian-Yu for chronic bacterial prostatitis in rats.

OBJECTIVES: Qian-Yu (QY), a Chinese medicine formula, has anti-inflammatory and broad spectrum anti-bacterial activity. This study is undertaken to evaluate the anti-inflammatory effects of Qian-Yu (QY) in chronic bacterial prostatitis (CBP).
MATERIALS AND METHODS: The potential of QY in the treatment of CBP was evaluated using a CBP animal model by examining the anti-inflammatory activity. Its consequences were analyzed by immunological and histopathological methods. Experimental chronic bacterial prostatitis was induced by instillation of bacterial suspension of Escherichia coli 7.5 × 10(5) CFU/ml into the prostatic urethra. Animals were followed up for four weeks and then treated with either 7 mg/kg QY or 7 mg/kg positive control agent-Qianlietai (QLT) or 1 ml of phosphate-buffered saline (PBS) for the controls. Ten rats in each group were sacrificed at the end of four weeks. The inhibition of inflammation and its consequences were analyzed histologically. Prostatic IL-8, SIgA and zinc concentrations were measured by ELISA, RIA and ICP-AES, respectively. The data was expressed as mean ± S.D. Least-significant difference (LSD) of one-way ANOVA (SPSS 12.0) was used to determine the differences of scores.
RESULTS: The histopathology showed resolving prostatitis in QY-and QLT-treated groups and the immunology showed reduction of IL-8 and increment of SIgA contents in prostatic tissues as compared to the control groups. The prostatic zinc levels were higher in the QY-and QLT-treated groups than those in the controls. These results suggested that QY is effective in CBP treatment.

Introduction

Chronic bacterial prostatitis (CBP) is one of the most common entities encountered in urological practice[1] and need long-term and high-dose antibacterial treatment.[2] However, antibacterial use is associated with side-effects and bacterial resistance. Hence, patients often seek herbal therapies outside of the traditional approaches.[3] Chinese medicinal formula QY has anti-inflammatory and broad-spectrum antibacterial effect.[4] It is widely used in China for the treatment of prostatitis and has produced a favorable effect.[5] The objective of this study is to investigate the pharmacodynamic of QY using a reproducible rat model of CBP.[6]

Materials and Methods

Preparation of Qian-Yu Formula

Qian-Yu formula was made of Radix Astragali, Epimedium Nakai., Leonurus heterophyllus Sweet, Cyathula of ficinalis Kuan, Scolopendra subspinipes mutilan, and Phellodendron chinens. It was prepared as described previously.[57]

Experimental Animals

Adult male Sprague-Dawley rats used in the study were of three to four months old, and weighed between 250 and 300 g at the start of the experiments. The animals had free access to food and water, and were allowed to acclimatize for at least one week before use. The study was approved by Institutional Animal Ethics Committee, Huazhong University of Science and Technology, China.

CBP Model

The CBP model of the rat was performed as previously described with a little modification.[6] A strain of Escherichia coli Z17 (02: K1: H-) was incubated overnight in tryptic soy broth (TSB) in a 37°C incubator. The E. coli cells were spun, washed three times and re-suspended in TSB to give 7.5 × 105 colony-forming units/ml (CFU/ml). Rats were anaesthetized with ether and the genital area was cleaned with 70% alcohol. Subsequently, the urethras of rats were catheterized with a sterile polyethylene tube (0.9 mm outer diameter, 2.5 cm length). Using an insulin syringe, 0.2 ml of the bacterial suspension (7.5 × 10 5 CFU/ml) was introduced into the prostatic urethra. Anesthesia was maintained for 1 h to allow a sufficient time for bacteria to invade the prostate. To confirm the establishment of CBP,[8] prostatic biopsy and urine sample collection were performed as follows: Four weeks after E. coli instillation, rats were anaesthetized with an intramuscular injection of a combination of ketamine (50 mg/kg) and xylazine (12 mg/kg). The ventral prostate gland was biopsied and urine was collected by cystocentesis for microbiological evaluation. Part of the prostate biopsy specimen and the urine sample were plated on MacConkey agar after dilution in phosphate-buffered saline (PBS). Rats showing CBP that was confirmed microbiologically (>20 CFU/g) and histologically (infiltration of inflammatory cells, interstitial fibrosis and obliteration of the acini) were selected for the study.

Experimental Groups and Treatments

The rats were divided into four groups of ten each. Group 1 was normal control group (n = 10) treated with 1 ml of phosphate-buffered saline (PBS, pH 7.2). Group II was CBP control (n = 10) received 1 ml of PBS (pH 7.2), group III was treated with QY (n = 10), 7 mg/kg body weight of QY dissolved in 1 ml of distilled water and group IV received Qianlietai (QLT) (n = 10), 7 mg/kg body weight dissolved in 1 ml of distilled water. All treatments were administered through an oral gavage tube once a day for four weeks.

Collection of Tissue Specimens and Samples

At the end of four weeks, all animals were anesthetized using a combination of ketamine (50 mg/kg) and xylazine (12 mg/kg). The surgical site was scrubbed with 10% povidone-iodine solution. A 2 cm long vertical midline abdominal incision was made and the abdominal muscle and peritoneum were incised. After the peritoneal cavity was exposed, the ventral prostate gland was excised carefully. Parts of the tissues were stored in 10% buffered formalin for pathological examination and the rests were washed by PBS and stored in deep freeze until for IL-8, SIgA and zinc level were estimated.

IL-8 Estimation

IL-8 level in the homogenate of prostate tissue was determined by the solid-phase enzyme-linked immunosorbent assay (ELISA) Quantikine IL-8 Immunoassay. The medium minimal detectable dose of the IL-8 assay was 10 pg/ml. All samples were tested in triplicate in agreement with the manufacturer's recommendations.

SIgA Estimation

SIgA levels in the homogenate of prostate tissue were detected by radioimmunoassay (RIA) using SIgA RIA kit. All samples were tested in triplicate as per the manufacturer's recommendations.

Zinc Estimation

Prostatic zinc levels were measured by ICP-AES at 213.856 nm. Ten normal rats of the same age without bacterial prostatitis were used for normal reference values of zinc in prostatic tissue. All samples were tested in triplicate.

Histological Study

Samples of the ventral prostate from each group were fixed in 10% buffered formalin, embedded in paraffin, sectioned into 5 mm thickness, stained with hematoxylin and eosin and examined using optical microscopy. The severity of prostatic inflammation was scored based on three parameters (the amount of chronic inflammatory cell infiltrates, acinar changes and interstitial fibrosis) in the most representative area by two pathologists who were blind to the treatment on the experimental group.

Statistical Analysis

The data was expressed as mean ± S.D. Least-significant difference (LSD) of one-way ANOVA (SPSS 12.0) was used to determine differences of scores. P < 0.05 was considered as statistical significant.

Results

IL-8 Level

For IL-8 assay, the regression equation was Y = -156.22 + 1653.34 X (r = 0.9960). Significant levels (P < 0.05) of IL-8 were found in QY-treated group compared to the CBP control group and no differences were observed between the QY- and QLT-treated groups [Table 1].

Table 1

Comparison of IL-8, SIgA and zinc levels in prostate tissues of rats treated with Chinese medicinal formula QY (n = 10 each group)

SIgA Level

For SIgA assay, the logarithm of regression equation was LOG (Y-Ysº) = 0.036 + 1.38LOG Cxi – 0.127(LOG Cxi)2(Ysº = 1801). Both QY and QLT groups were significant (P < 0.05) as compared to control group (P < 0.05). However, no differences were observed between the QY and QLT groups [Table 1].

Zinc Concentrations in Prostate

Zinc levels were significantly decreased (P < 0.05) in rats with chronic bacterial prostatitis as compared to normal control group. There was a significant (P < 0.05) increase in prostatic zinc concentration at four weeks in QY- and QLT-treated groups as compared tothe CBP controls [Table 1].

Histopathological Data

Prostate section of normal control rats is shown in Figure 1. Intense infiltration of lymphocytes into glandular acini and interstitium was observed in the infected prostates of the CBP control [Figure 2]. This chronic inflammatory change led the acini to undergo obliterative change and atrophy. In QY-treated group and QLT-treated group at week 4, the infiltration of inflammatory cells had decreased in both the glandular acini and interstitium tissues and normal to minimally inflamed glands were observed in most areas. Acini were restored in size and their lumen contained few lymphocytes. Stroma showed mild fibrous tissue proliferation and comparatively normal appearance [Figures 3 and 4].

Figure 1

Prostate section of normal prostatitis rat

Figure 2

Prostate section of chronic bacterial prostatitis rat obtained four weeks after phosphate-buffered saline (control) treatment. The acinar structures were severely atrophied and obliterated. Marked chronic inflammatory cell infi ltration and interstitial fi brosis were seen

Figure 3

Prostate section of chronic bacterial prostatitis rat obtained four weeks after QY treatment. The acinar structures were mildly atrophied and obliterated. Mildly chronic infl ammatory cell infi ltration and interstitial fi brosis were seen

Figure 4

Prostate section of chronic bacterial prostatitis rat obtained four weeks after QLT treatment. The acinar structures were mildly shrunken. Mild lymphocytic infi ltration and fi brosis in the interstitial space were seen

Discussion

CBP is a chronic inflammatory process linked with changes in the immune system.[9] IL-8 is a pro-inflammatory cytokine and was synthesized and secreted by a variety of somatic cells and could be detected after a few hours of infection. Some researches showed that IL-8 was detected in a high proportion of men with CP/CPPS but was undetectable in normal people, confirming the role of this chemokine in the pathogenesis of CP.[10] Secretory IgA (SIgA) is the primary mediator of immunity in mucosal areas. Its function is to maintain a balance between immunological responses against foreign pathogens. Therefore, it is served as a first line of defense in mucosal areas by inhibiting adhesion of microorganisms.[11] Based on the findings mentioned above, we therefore suggested that the contents of SIgA and IL-8 in prostate tissues could be considered as the optimal diagnostic markers.

Zinc is a well-known antibacterial factor trace and insufficient intake manifests severe immune impairment and infections in human.[12] High concentration of zinc has been found in prostate.[13] Many investigators have found that chronic prostatitis is associated with a decrease in the zinc level.[14] Thus, patients with chronic prostatitis are susceptible to recurrent infection. Hence, maintaining the zinc at normal level would be helpful for the CBP treatment.

Chinese medicinal formula QY is widely used in China for the treatment of CBP and produced a favorable effect. A large body of experimental data suggested that Radix Astragali, Leonurus heterophyllus Sweet, and Scolopendra subspinipes mutilan, the constitutions of QY, were proven to be responsible for antimicrobial and anti-inflammatory activities. It has been reported that the flavonoids in Radix Astragali,[15] the alkaloids in Leonurus heterophyllus Sweet[16] and the antibacterial peptide in Scolopendra subspinipes mutilan[17] contribute to these effects.

The present study showed significant therapeutic effects of QY by influencing the IL-8, SIgA and zinc levels and could be used for CBP treatments. However, the real bioactive compounds are still unknown. Thus, it is essential to determine the real bioactive compounds of QY for controlling the quality and safeguarding the public. Therefore, further investigation is required for this promising Chinese medicinal formula.