Reduced corporal fibrosis to protect erectile function by inhibiting the Rho-kinase/LIM-kinase/cofilin pathway in the aged transgenic rat harboring human tissue kallikrein 1

Abstract

Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR) harboring the human tissue kallikrein 1 (hKLK1), while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED) is poorly understood. Male wild-type Sprague-Dawley rats (WTR) and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR), and aged TGR (aTGR). Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson's trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson's trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC)/collagen (C) was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-β 1 (TGF-β1), RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED.

Figure 1

Erectile function of all rats was measured through electric stimulation on the cavernous nerves. Representative carotid artery pressure and ICP tracing were measured through stimulation of 2.5 V (a) and 5 V (b) setting for 1 min, respectively, in rats of all groups. The max ICP/MAP ratio of different volts were presented through bar graphs: (c) for 2.5V, and (d) for 5V. Data are expressed as mean ± s.d. (n = 10 rats per group). *P < 0.05 when comparing the two groups under each end of the capped line. ICP: intracavernous pressure; MAP: mean arterial pressure; yWTR: young wild-type Sprague-Dawley rats; aWTR: aged wild-type Sprague-Dawley rats; aTGR: aged transgenic rats; s.d.: standard deviation.

Figure 2

Verification of expressions of hKLK1 and rKLK1 genes in the corpus cavernosum. (a) Representative hKLK1 genomics DNA bands in rats’ corpus cavernosum by agarose gel electrophoresis followed by conventional PCR. Relative mRNA expressions of rKLK1 (b) and hKLK1 (c) with β-actin as the loading control in the corpus cavernosum of three groups by real-time RT-PCR. (d) Representative Western blotting results of rKLK1, hKLK1, and β-actin in the corpus cavernosum of three groups. Expressions of rKLK1 (e) and hKLK1 (f) with β-actin as the loading control in the corpus cavernosum of three groups were presented through bar graphs. Data are expressed as mean ± s.d. (n = 10 rats per group). *P < 0.05 when comparing the two groups under each end of the capped line. RT-PCR: reverse transcriptase PCR.

Figure 3

Effects of hKLK1 on histological changes of penile tissues. (a) Masson's trichrome staining (×200) of corpus cavernosum: the area of smooth muscle is represented by red stain and the area of collagen is blue stain. Expressions of α-SMA (b) and TGF-β1 (c) were assessed through immunohistochemistry (×200). (d) Representative Western blotting results of α-SMA, TGF-β1 and β-actin in the corpus cavernosum of three groups. Expressions of α-SMA (e) and TGF-β1 (f) with β-actin as the loading control in the corpus cavernosum of three groups were presented through bar graphs. (g) The ratio of smooth muscle/collagen. Data are expressed as mean ± s.d. (n = 10 rats per group). *P < 0.05 when comparing the two groups under each end of the capped line. yWTR: young wild-type Sprague-Dawley rats; aWTR: aged wild-type Sprague-Dawley rats; aTGR: aged transgenic rats; s.d.: standard deviation

Figure 4

Immunohistochemistry and Western blotting analysis of the activation of RhoA/ROCK pathway. Expressions of RhoA (a) and ROCK1 (b) were assessed through immunohistochemistry (×200). (c) Representative Western blotting results of RhoA, ROCK1, p-MYPT1 and β-actin in the corpus cavernosum of three groups. Expressions of RhoA (d), ROCK1 (e) and p-MYPT1 (f) with β-actin as the loading control in the corpus cavernosum of three groups were presented through bar graphs. Data are expressed as mean ± s.d. (n = 10 rats per group). *P < 0.05 when comparing the two groups under each end of the capped line. yWTR: young wild-type Sprague-Dawley rats; aWTR: aged wild-type Sprague-Dawley rats; aTGR: aged transgenic rats; s.d.: standard deviation.

Figure 5

Immunohistochemistry and Western blotting analysis of the activation of LIMK2/cofilin pathway. Expressions of p-LIMK2 (a) and p-cofilin (b) were assessed through immunohistochemistry (×200). (c) Representative Western blotting results of LIMK2, p-LIMK2, cofilin, p-cofilin, and β-actin in the corpus cavernosum of three groups. Expressions of LIMK2 (d), p-LIMK2 (e), cofilin (f), and p-cofilin (g) with β-actin as the loading control in the corpus cavernosum of three groups were presented through bar graphs. Data are expressed as mean ± s.d. (n = 10 rats per group). *P < 0.05 when comparing the two groups under each end of the capped line. s.d.: standard deviation.