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. 2022 Mar 20;42(3):411-417.
doi: 10.12122/j.issn.1673-4254.2022.03.14.

[Pirfenidone alleviates urethral stricture following urethral injury in rats by suppressing TGF-β1 signaling and inflammatory response]

[Article in Chinese]
Z Li  1 , X Huang  2 , S Chen  1 , Z Zhang  2 , X Liang  1 , H Li  3 , L Qin  4 , Y Guo  1
Affiliations

[Pirfenidone alleviates urethral stricture following urethral injury in rats by suppressing TGF-β1 signaling and inflammatory response]

[Article in Chinese]
Z Li et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the effect of pirfenidone for reducing urethral stricture following urethral injury in rats and explore the possible mechanism.

Methods: Thirty male SD rats were randomly assigned into negative control group, positive control group and pirfenidone group (n=10). In pirfenidone and positive control groups, the rats were subjected to incision of the posterior urethral cavernous body followed by daily intraperitoneal injection of pirfenidone (100 mg/kg) and an equivalent volume of solvent, respectively. The rats in the negative control group were given intraperitoneal injections of solvent without urethral injury. At two weeks after modeling, retrograde urethrography was performed for observing urethral stricture, and the injured urethral tissues were harvested for HE staining, Masson staining, immunohistochemical staining and Western blotting for detecting the protein expressions of α-SMA and TGF-β1. The mRNA expressions of the inflammatory factors TNF-α, IL-6, and IL-1β were detected using qRT-PCR.

Results: The body weight of the rats in pirfenidone group was significantly decreased compared with that in the other two groups (P < 0.05). Retrograde urethrography showed significant narrowing of the urethra in the positive control group but not in the pirfenidone group. HE staining of the injured urethral tissues showed obvious proliferation of urethral epithelial cells with narrow urethral cavity and increased inflammatory cells in positive control group. The pathological findings of the urethra were similar between pirfenidone group and the negative control group. Masson staining revealed obviously reduced collagen fibers and regular arrangement of the fibers in pirfenidone group as compared to the positive control group. Compared with those in the negative control group, the expressions of α-SMA and TGF-β1 were significantly increased in the positive control group, and pirfenidone treatment significantly inhibited their expressions (P < 0.05 or 0.01). Pirfenidone also significantly inhibited the mRNA expressions of TNF-α, IL-6, and IL-1β in the injured urethral tissue (P < 0.05 or 0.01).

Conclusion: Pirfenidone can prevent urethral fibrosis and stricture after urethral injury possibly by inhibiting the TGF-β1 pathway and inflammatory response.

目的: 探讨吡非尼酮对大鼠尿道损伤后狭窄形成的预防作用及可能机制。

方法: 选取SD雄性大鼠30只，随机分为3组（10只/组）：阴性对照组、阳性对照组及吡非尼酮组。吡非尼酮组：切开后尿道海绵体建立大鼠尿道损伤模型，按100 mg·kg-1·d-1腹腔注射吡非尼酮；模型组：同对照组构建大鼠尿道损伤模型，腹腔注射等量溶剂；假手术组：不予尿道损伤处理，但腹腔注射等量溶剂。术后2周逆行尿道造影观察尿道狭窄，留取尿道损伤组织，行HE染色观察尿道组织形态学变化，Masson染色检测胶原变化，免疫组化及Western blot检测a-SMA和TGF-β1的蛋白表达，qRT-PCR检测大鼠尿道组织中炎症因子TNF-α、IL-6、IL-1β mRNA的表达。

结果: 吡非尼酮组大鼠较阴性对照和阳性对照组体质量下降，逆行尿道造影显示阳性对照组大鼠尿道显著变窄，吡非尼酮组大鼠尿道较阳性对照组大鼠明显好转（P < 0.05）。HE染色显示阳性对照组尿道上皮细胞增生，管腔狭窄，炎性细胞增多；吡非尼酮组病理学表现与阴性对照组相似。Masson染色显示吡非尼酮组较阳性对照组胶原纤维含量少，排列规则有序。免疫组化和Western blot结果表明阳性对照组尿道a-SMA和TGF-β1表达显著高于阴性对照组（P < 0.05，P < 0.01），而吡非尼酮能够抑制TGF-β1和α-SMA的表达。qRT-PCR结果显示吡非尼酮能够抑制损伤组织中TNF-α、IL-6、IL-1β等炎症因子的基因表达（P < 0.05，P < 0.01）。

结论: 吡非尼酮可预防尿道损伤后纤维化及狭窄，并可能与抑制TGF-β1通路和炎症反应相关。

Keywords: fibrosis; inflammation; pirfenidone; transforming growth factor-β1; urethra stricture.

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Figures

图 1
图 1
大鼠尿道损伤模型的构建 Establishment of urethral injury model in rats. A: Normal urethra. B: Urethral dissection. C: Placement of the indwelling needle into the urethra. D: Suture of the urethral sponge. E: Reconstruction of the external orifice of the urethra. F: Healed urethra.
图 2
图 2
3组大鼠的体质量增长情况 Weight changes of the rats in the 3 groups. *P < 0.05 vs negative control, #P < 0.05 vs positive control group.
图 3
图 3
3组大鼠逆行尿道造影 Retrograde urethrography of the rats in the 3 groups. A: Negative control group. B: Positive control group. C: Pirfenidone group.
图 4
图 4
3组大鼠尿道组织HE染色结果 HE staining of the urethral tissues in the 3 groups (Original magnification: × 100). A: Negative control group. B: Positive control group. C: Pirfenidone group.
图 5
图 5
3组大鼠尿道组织狭窄比较 Comparison of urethral stricture in the 3 groups. A-C: HE staining (×100). D-F: Masson staining (×100). G-I: Immunohistochemical staining for α-SMA (×200). J-K: Quantitative analysis of Masson (J) and α-SMA (K) immunohistochemical staining results. *P < 0.05, **P < 0.01 vs negative control, #P < 0.05, ##P < 0.01 vs positive control.
图 6
图 6
3组大鼠尿道组织TGF-β1的蛋白表达 Expressions of TGF-β1 in the urethral tissues. A-C: Expressions of TGF-β1 detected by immunohistochemical staining in negative control group (A), positive control group (B), and pirfenidone group (C) (× 200). D. Quantitative analysis of immunohistochemical staining results. E: Western blotting for detecting the expression of TGF-β1. F-G: Quantitative analysis of Western blots. *P < 0.05, **P < 0.01 vs negative control, #P < 0.05, ##P < 0.01 vs positive control.
图 7
图 7
qRT-PCR检测TNF-α、IL-6、IL-1β炎症因子的表达 Expressions of TNF-α, IL-6 and IL-1β mRNA detected by qRT-PCR. **P < 0.01 vs negative control, #P < 0.05, ##P < 0.01 vs positive control.
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