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ORIGINAL ARTICLE
Year : 2022 | Volume
: 33
| Issue : 4 | Page : 205-212
The effect of intraurethrally applied anatolian propolis extract on urethral healing in a rat model
Reha Girgin1, Emine Yilmaz Can2, Gökhan Çeker3, Esin Kaymaz4, önder Çinar1, Necmettin Aydin Mungan1
1 Department of Urology, School of Medicine, Zonguldak Bulent Ecevit University, Zonguldak, Turkey
2 Department of Pharmacology, School of Medicine, Zonguldak Bulent Ecevit University, Zonguldak, Turkey
3 Department of Urology, Samsun Vezirköprü State Hospital, Samsun, Turkey
4 Department of Pathology, Dr. Ridvan Ege Hospital, Ufuk University, Ankara, Turkey
Correspondence Address:
Reha Girgin
Department of Urology, Medical Faculty, Zonguldak Bulent Ecevit University, Zonguldak
Turkey
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/UROS.UROS_158_21
Purpose: Urethral stricture may cause irreversible results, as it prevents normal voiding. Although various endoscopic and open surgical options are available, the results are not always satisfactory so the main purpose is to prevent the formation of urethral scar. Our purpose was to examine the effects of intraurethrally administered Anatolian propolis on healing after an experimental urethral injury. Materials and Methods: A total of 40 Wistar male rats were used. Rats were divided into five equal groups: healthy control (Group 1), urethral damage/pathology (Group 2), solvent control (Group 3), 1-week propolis treatment (Group 4) and 3-week propolis treatment (Group 5). Urethral damage was performed with a 29G needle. Intraurethral, 50% ethanol, was administered in the solvent control group and 30% propolis was administered intraurethrally to the rats in Groups 3 and 4. Penile tissues were taken under deep anesthesia and examined under a light microscope. Results: Irregularities and luminal narrowing in the urethral epithelium and connective tissue were found in Group 2, except for one rat. Similarly, hyperemia-bleeding was observed in all rats except for one rat. Irregularities and hyperemia in the urethral epithelium and connective tissue were found in Group 3, except for two rats. Total improvement was observed in one rat, and more than 50% of fibrosis was observed in four rats in Groups 2 and 3. In Group 4, irregularity was observed in the urethral epithelium in four rats, while no inflammation was found in five rats. All of the rats had <50% fibrosis. In Group 5, six rats had complete recovery and <30% fibrosis. Conclusion: Anatolian propolis applied into the urethra seems to accelerate recovery after urethral trauma and reduces the formation of fibrosis.
Keywords: Anatolian propolis, intraurethral, urethral healing, urethral stricture
Urethral stricture is described as the narrowing of the urethra as a result of scar tissue formation due to the increased fibroblastic activity of the spongy corpus spongiosum.[1] It may cause irreversible results as it prevents normal voiding.[2] Although various endoscopic and open surgical options are recommended as a treatment approach, the results are not always satisfactory.[3] Because the main purpose is to prevent the formation of urethral scar tissue, many oral and intraurethral agents have been investigated in this context.[4],[5]
Wounds following tissue injuries heal, and the healing process begins with an inflammatory response and ends with scar formation.[1] During this complex process, the main role is played by the fibroblast through the effect of various cytokines and growth factors.[1] This results in the accumulation of large amounts of matrix components, such as collagen and glycosaminoglycan, in the wound site.[6],[7]
Excessive extracellular matrix (ECM) accumulation determines the prognosis of the urethral healing process, and it is aimed to be corrected in the treatment. Although ECM proteins are among the main components of normal tissues, there are significant changes in the levels and types within the scar tissue.[6]
Propolis, a bee product, has many biological activities.[8] Therefore, it has an area of application in medical science in the treatment of various skin, rheumatic, heart diseases, and cancer.[8] In light of this information, we aimed to evaluate the histopathological effects, including fibrotic changes of intraurethrally administered propolis, on urethral wound healing in rats.
Materials and MethodsThis study was conducted in the laboratory of Zonguldak Bülent Ecevit University (protocol number: 2019-07-04/04) after obtaining approval from the local ethics committee. A total of 40 Wistar albino male rats (200–252 g) were anesthetized by intraperitoneally administering a combination of 90 mg/kg ketamine (Ketalar-Eczacibaşi, Turkey) and 10 mg/kg xylazine (Rompun-Bayer, Turkey). Experiments were conducted in five groups [Table 1]: healthy control (Group 1), urethral damage/pathology (Group 2), 3-week solvent control (Group 3), and 1-week (Group 4) and 3-week (Group 5) urethral propolis treatment groups.
Urethral injury was induced as previously described by Ayyildiz et al.[1] The damage was created by scratching the ventral surface of the urethral epithelium after the insertion of a 29-g syringe tip approximately 5 mm from the external meatus after giving it a hook shape. Experimental urethral damage was induced in all rats other than the healthy control group. Following urethral injury, 50 μL of 50% ethanol was administered to the rats in Group 3 by inserting a plastic-tipped angiocath into the urethra for 3 weeks daily. Group 4 was administered 20 mg (50 μL) of Anatolian propolis once a day for 1 week and Group 5 was administered 20 mg (50 μL) once a day for 3 weeks by inserting a plastic-tipped angiocath into the urethra. The biochemical content of the propolis we used is presented in [Table 2].
Table 2: Features of intraurethrally administered propolis in study groupsIn this study, a commercially produced BEEO UP 30% propolis solution (SBS Bilimsel Bio Çözümler San ve Tic. A.Ş. -SBS Bilimsel Bio Çözümler R&D Centre [Bee'o /Bee&You R&D Centre]/Turkey) was used. Anatolian propolis was collected from beehives in different regions in Turkey and confirmed by an agricultural organization. In general, after the collection, propolis is mixed with beeswax and must be extracted to make it ready for use. Although many extraction methods can be used for propolis, the most preferred one is by dissolving it with ethyl alcohol. In the experiment, the main components of propolis were cut into small pieces, mixed with 250 mL of 70% ethanol (25 g), and incubated at room temperature for 48 h with shaking (150 rpm).
The extract obtained was purified with Whatman filter paper, and the ethanol content was evaporated using a rotary vacuum evaporator to obtain a purified propolis extract. Then, the purified extract was diluted using 50% ethanol to obtain a 30% propolis solution. The extracts were stored at 2°C–8°C under protective-light conditions and warmed to room temperature just before injection.
Because no substance was given to the rats in Groups 1 and 2, they provided the baseline histopathological data. The rats were kept in their special cages in rooms with a constant room temperature (22°C–24°C), humidity (50%–55%), and light/dark cycle (12-h light/12-h dark). Water and feed were regularly changed. A cage base consisting of sterilized dust-free wood shavings changed three times a week was used. The rats were sacrificed at the end of the 3rd week by administering high-dose anesthesia. All rats were penectomized from the proximal injured area, and the tissues were placed in 10% formaldehyde solution separately.
Histopathological evaluation
After fixation at the appropriate time, each group was coded separately and taken for macroscopic examination and sampling. Penectomy materials were examined by slicing in the transverse axis. Two circular samples were obtained from the meatus and middle part, with urticaria passing through it. The samples were coded and blocked separately and followed routinely. After the paraffin-embedding procedures, two sections, each 4-μm thick, were taken from each sample. One of these sections was stained with hematoxylin-eosin dye for routine examination. The other section was stained with Masson's trichrome histochemical stain to evaluate fibrosis. Staining with Masson's trichrome was performed automatically using a Ventana Roche BenchMark Special Stain histochemistry device. The materials were examined using a light microscope.
The presence of hyperemia–hemorrhage in the corpus spongiosum, as well as inflammation, was evaluated in the hematoxylin–eosin-stained sections. Furthermore, urethral narrowing and the presence of papillary structures in the epithelium were evaluated. Finally, it was evaluated whether full recovery could be achieved. Hyperemia–hemorrhage and urethral narrowing in the corpus spongiosum and evaluation of papillary structures in the epithelium were performed in the form of present or absent.
We considered the inflammatory cells localized to the lamina propria under the epithelium for the evaluation of inflammation.[9] Primarily, we classified the inflammatory cells as acute or chronic according to the predominance of neutrophils or lymphoplasmocytic cells, respectively.[9] Subsequently, we graded the inflammatory cells according to their density as mild, moderate, and severe. In this evaluation, we counted the number of cells per mm2. Accordingly, we considered the presence of 5–25 lymphocytic cells per mm2 only in the lamina propria as mild. If inflammatory cells infiltrate the epithelium, we classified it as moderate or severe. The presence of 26-100 lymphocytic cells per mm2 was assumed as moderate and more than 100 lymphocytic cells per mm2 as severe.
For the histochemical evaluation, four large magnification areas (×40) were randomly scanned in the penile corpus spongiosum area in each section stained with Masson's trichrome, and the ratio of fibrosis and collagen tissue to muscle tissue was calculated. The blue and red areas represent the collagen and Masson's trichrome-stained muscle tissue, respectively. Quantitative analysis of the collagen areas was conducted using the ImageJ software, an open-source freeware (U.S. National Institutes of Health, USA; website: http://rsb.info. nih.gov/ij/download.html). Accordingly, fibrosis was graded in four groups according to the density of the amount of collagen.
Statistical analysis
Statistical analysis was conducted using R-4.0.2. The descriptive statistics of the qualitative variables are expressed in frequency and percentage. Fisher–Freeman–Halton and Fisher's exact Chi-squared tests were employed for the intergroup comparisons of the variables. In all statistical comparisons in the study, results with a P < 0.05 were considered statistically significant.
ResultsInflammatory changes were in the form of chronic inflammation in all groups. When the groups were compared in terms of inflammation, the group with the least inflammatory changes was the first group of healthy rats. In this group, only mild inflammation was observed, which occurred in only one rat. In Group 2, intense inflammation was detected as expected (severe in three rats, moderate in three rats). Although the inflammation observed in Group 3 was not as severe as the pathology group, it was generally characterized by moderate inflammation (moderate to mild in seven rats). Groups 4 and 5 were similar in terms of inflammation findings, i.e., a significant reduction in inflammation (no inflammation in five and six rats, respectively). The intensity of inflammation in Group 2 was statistically different from those in Groups 4 and 5. The inflammation findings in all groups are presented in [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e.
Figure 1: Evaluation of inflammation in subepithelial tissue and corpus cavernosum, (a) No inflammation was observed in the healthy group marked with an arrow (H and E, × 400), (b) In this image, periurethral, subepithelial area has severe inflammation. Note that the inflammatory cells are more than 100 in one HPF and also some of them attack the epithelium (H and E, × 400). These inflammatory cells have been demonstrated with an arrow. This image belongs to the pathology group, (c) Note that there are inflammatory cells in the subepithelial region. This inflammation is not severe as figüre 1b and also not weak as in image 1d. This image is an example of moderate inflammation (H and E, × 400), (d and e) These are similar images in terms of the density of inflammatory cells. Note the sparse and scattered cells that are marked with an arrow. The count of them is < 5 for one HPF. This density of inflammation belonged to Groups 4 and 5 and has been graded as “low” (H and E, × 400). HPE: High power fieldIn our study, the most important indicator of urethral epithelial damage was the narrowing of the urethral lumen and the presence of papillary structures. Spongiotic damage was compared between the groups in terms of the presence of hyperemia. From this viewpoint, none of these findings was detected in the rats in Group 1, whereas in the majority of the rats in Groups 2 and 3, these findings indicating damage were observed. Narrow urethral lumen, papillary structures, and hyperemia were significantly reduced in Group 5. These findings were found to be statistically significant between Groups 2 and 5. Although a difference between Groups 2 and 4 was observed, these findings were not statistically significant. [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e presents the prominent histomorphological findings in each group. Therefore, total healing, which we evaluated as the disappearance of the aforementioned findings, was statistically significant between Groups 2 and 5.
Figure 2: Evaluation of hyperemia and healing of the urethra, (a) In the healthy control group, the open urethral lumen and corpus cavernosum without hyperemia (H and E, × 200), (b and c) These two images demonstrate the pathology and solvent control group. Both of them are similar in terms of hyperemia and healing of the urethra. Note that the significant bleeding in the subepithelial area in a rat of Group 2 (marked with a blue arrow) and narrowing of the urethral lümen-prominent papillary configurations like fingers in the epithelium in a rat of Group 3 (marked with a black arrow) (H and E, × 200), (d and e) These images belong to Groups 4 and 5. In these images, there is not significant hyperemia. And also, the lumen of the urethelium is open like normal urethelium, as seen in image 2a (H and E, × 200)A comparison of the groups in terms of fibrosis was performed. Although differences were observed in the comparison, they were not statistically significant. The degrees of fibrosis are presented in [Figure 3]. Fibrosis was detected at <30% in all rats in the healthy control group [Figure 3]a but increased in half of the rats in Groups 2 and 3 (50%–70% or >70%) [Figure 3]b and [Figure 3]c. No significant fibrosis was detected in any of the rats in Groups 4 [Figure 3]d and 5. In Group 5, fibrosis was detected at <30% in six rats, as observed in healthy rats [Figure 3e]. However, this finding did not exhibit statistical significance.
Figure 3: Grading of fibrosis with trichrome stain. For this stain, the blue color represents the collagen. The arrow in each image points to collagen, (a) Note the percentage of blue color in this image is low, nearly 30% (×400), (b) >70% fibrosis (×400) in a case belonging to the study group. In this image, the intense blue color means fibrosis has markedly increased, (c) In this picture, we see the intense blue color again (×400). When the collagen represented by the blue color is compared to the general, it was determined to be on the scale of 50%–70%, (d and e) These images belong to Groups 4 and 5. In these images, note that the collagen is not as dense as in b and c Fibrosis was 30%–50% (×400)All histopathological findings in the groups are summarized in [Table 3].
DiscussionIncreased urethral scar tissue resulting in decreased urine flow is the most catastrophic result of urethral injury. At present, surgeries performed by removing the scar tissue and replacing it with native tissue yielded the most satisfactory results.[10] However, because such surgeries are complex, endoscopic surgeries are preferred, the results of which are not satisfactory at all.[10] In this process, in which patients often go into a vicious circle, the main goal should be to prevent the formation of scar tissue.
Urethral healing occurs in four stages, similar to dermal healing, and is completed with the remodeling phase that determines the final appearance of the tissue.[11],[12] The wound healing process can be negatively affected by infection, insufficient oxygenation, tissue nutrition, and oxidative stress factors.[12] Despite the current knowledge on tissue healing, there is no available treatment that can accelerate this process.[12] There are, however, studies on various oral and intraurethral agents to prevent this process. Agents such as intraurethral triamcinolone, hydrocortisone–furacine combination, and honey have been used in clinical studies. In addition, various growth factors, chemotactic agents, and gene carriers have been experimentally studied.[1],[13],[14]
At present, natural products have found increasing interest and use in modern disease management. In this regard, bee products, in particular, have attracted attention owing to their being natural and ancient. Propolis is a resinous product produced by bees in nature and used while constructing their hives by sealing them to protect new generations against pathogenic organisms.[15] In the chemical structure of propolis, around 300 compounds, including various amino acids, vitamins, fats, and carbohydrates, have been identified.[16],[17] However, phenols, including phenolic acid and flavonoids, constitute the main active group.[17] Many studies have reported that propolis has antimicrobial, anti-inflammatory, antioxidant, antiulcer, anticancer, and cardio-, hepato-, and neuroprotective effects.[17],[18] In addition, it has been shown to be effective in wound healing in many studies.[12],[18],[19],[20],[21],[22] However, to the best of our knowledge, our study is the first to investigate the protective effects of propolis against urethral damage.
Fibrosis is defined as an excessive accumulation of fibrous connective tissues (ECM components such as collagen and fibronectin) in and around damaged tissues, which can lead to permanent scarring and loss of function in the affected tissue.[23] The inflammatory process that is known to play a key role in wound healing is also associated with scar formation.[24] Although inflammatory cells such as neutrophils and monocytes are important in the removal of necrotic tissue and bacteria from the area of benefit, the reactive oxygen radicals they release can paradoxically cause damage to the environment.[23] As a result, if inflammatory macrophages and neutrophils are not immediately eliminated, they can worsen the inflammatory response that causes scarring and tissue damage.[25] This is best evidenced by the fetal wound healing without a scar due to the absence of the typical inflammatory response.[26] Hormonal changes that reduce the inflammatory response will also reduce scar formation.[26]
The effects of propolis on wound healing are thought to be caused by its anti-inflammatory and antibacterial activities.[19] Propolis inhibits the activity of cyclooxygenase (COX) and lipoxygenase, thereby reducing the production of prostaglandin E2 and the expression of the inducible isoform of COX-2.[24] Furthermore, it decreases neutrophils and macrophages by downregulating the protein expression of the inflammatory transcription factor nuclear factor kappa-light-chain enhancer of activated B-cells (pNF-κB) and reducing the production of tumor growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), and interleukin-6.[23],[24] In terms of its antibacterial effects, previous research suggested that propolis inhibits lipid metabolism, thereby affecting membrane stability and leading to cell lysis.[27],[28] Studies also demonstrated that it inhibits bacterial ribonucleic acid polymerase and interferes with bacterial motility, cell division, and protein synthesis.[27],[28]
In addition, studies have shown that propolis reduces the ECM protein fibronectin responsible for collagen fibrosis.[19],[29] Propolis caused changes in the composition of the ECM and reduced ECM destruction by decreasing TNF-a-induced matrix metalloproteinases and changing the ratio of collagen type 1–3 in favor of type 1.[30],[31],[32],[33] Propolis triggers wound healing by increasing the expression of TGF-β, which plays a key role in wound healing by maintaining balanced excess fibroblastic activity by decreasing cells that secrete TGF-β.[19],[34] Moreover, propolis is thought to facilitate the cell repair process during wound healing by decreasing inflammatory cell activity and reducing free-radical activity.[12],[19]
In our study, we demonstrated the anti-inflammatory effect of propolis, which we applied intraurethrally, in Groups 4 and 5. We found that inflammation decreased more in Group 5 than in Group 4, and thus, its anti-inflammatory activity increased over time. The healing process was 75% complete in Group 5. Furthermore, we saw that the antifibrotic efficacy reached 75% in Group 5.
The effects associated with the topical use of ethanol have been evaluated in the literature, and although it has been emphasized that it may cause local irritation and allergic contact dermatitis, there have been conflicting results.[35] A low-toxicity profile has been reported in the local use of hydroalcoholic propolis, in which ethanol was used as a solvent.[36],[37],[38] In our study, it was observed that alcohol administration in the solvent group did not increase the pathological damage compared with the pathology group. Therefore, it can be deduced that the effect of alcohol as a solvent can be ignored.
Propolis is a product with an extremely rich biological content.[39] Among these biological contents, flavonoids and phenolic compounds are especially responsible for their antimicrobial, anti-inflammatory, and wound healing properties.[12] Flavonoids have been scientifically shown to reduce lipid peroxidation. At present, any drug that reduces lipid peroxidation is believed to reduce cell damage and increase cell synthesis.[40] Furthermore, flavonoids and terpenoids are responsible for promoting the healing process of injuries owing to their astringent and antimicrobial properties.[40]
ConclusionsAnatolian propolis, which we applied to the urethra, was found to have the ability to reduce inflammation and prevent the development of fibrosis because of urethral damage. At present, the increasing interest in propolis has also increased its application. Unlike other bee products, propolis, with its multiple mechanisms of action, is a natural product that has the potential to prevent urethral scarring and narrowing when administered intraurethrally. However, more studies are needed to describe this issue better.
Acknowledgments
We thank to “SBS Bilimsel Bio Çözümler San ve Tic. A.Ş” for their effort to provide the Anatolian propolis used in our study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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