ATP合酶在糖尿病大鼠阴茎海绵体平滑肌细胞病变中的作用机制

The role of ATP synthase in pathological changes of penile cavernous smooth muscle cells in diabetic rats

目的探讨三磷酸腺苷(ATP)合酶在糖尿病大鼠阴茎海绵体平滑肌细胞病变中的作用机制。方法体内实验:40只SD大鼠随机分为正常对照组与糖尿病组(按60mg/kg腹腔内注射链脲佐菌素后72h、1周、2周随机血糖水平>16.7mmol/L为造模成功),饲养10周后比较大鼠阴茎勃起功能;处死后取阴茎海绵体组织,采用胶原纤维染色法检测纤维化程度,免疫组化法检测Caspase-3表达,Western blot法检测F1-ATPase蛋白表达。体外实验:取正常大鼠阴茎海绵体平滑肌细胞进行细胞培养,分为对照组(正常培养基培养)、高糖组(含30mmol/L葡萄糖的培养基培养)、高糖+ATP酶过表达组(ATPase高表达质粒转染阴茎海绵体平滑肌细胞24h后,再用含30mmol/L葡萄糖的培养基培养)。48h后采用Western blot法检测F1-ATPase、转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)蛋白表达。结果在体内实验中,与正常对照组比较,糖尿病组30min内阴茎勃起次数明显减少(P<0.05),阴茎海绵体胶原纤维/肌纤维比例明显增加(P<0.05),Caspase-3阳性的平滑肌细胞比例明显升高(P<0.05),阴茎海绵体F1-ATPase蛋白表达明显降低(P<0.05)。在体外实验中,与高糖组比较,高糖+ATP酶过表达组F1-ATPase蛋白水平较高糖组明显升高(P<0.05),CTGF、TGF-β1蛋白表达明显降低(P<0.05)。结论高糖状态会导致阴茎海绵体纤维化,促进阴茎海绵体平滑肌细胞凋亡。通过线粒体ATP酶的上调,可以抑制TGF-β1途径,改善阴茎海绵体平滑肌纤维化程度,恢复平滑肌的功能,促使阴茎勃起功能得到改善。

Objective To investigate the role of adenine nucleoside triphosphate(ATP)synthase in the pathological changes of penile cavernous smooth muscle cells in diabetic rats.Methods Forty SD rats were randomly divided into normal control group and diabetic group.The diabetic model was induced by intraperitoneal injection of 60 mg/kg streptozotocin.The penile erectile function was observed and compared between two groups after 10 w of feeding.The blood and penile tissue samples were collected after the animals were sacrificed.The degree of fibrosis of the penile corpus cavernosum was detected by collagen fiber staining,Caspase-3 expression was detected by immunohistochemistry,and F1-ATPase expression was detected by Western blot.In vitro experiments the penile cavernous smooth muscle cells were cultured.The cells were divided into control group,high glucose group(medium containing glucose 30 mmol/L)and high glucose+ATPase over expression group.After 48 h of cell culture,the expression of F1-ATPase,transforming growth factor-β1(TGF-β1)and connective tissue growth factor(CTGF)were detected by Western blot.Results In vivo experiments,compared with the normal control group,the number of erections in diabetic group decreased significantly(P<0.05),the ratio of collagen fibers to muscle fibers in the corpus cavernosum increased significantly(P<0.05),the proportion of smooth muscle cells positive for Caspase-3 increased significantly(P<0.05),and the expression of F1-ATPase protein in the corpus cavernosum decreased significantly(P<0.05).In vitro experiments,the F1-ATPase protein level in the high glucose+ATPase over expression group was significantly higher than that in the high glucose group(P<0.05),while the expression of CTGF and TGF-β1 protein was significantly lower(P<0.05).Conclusion High glucose status can lead to fibrosis of penile corpus cavernosum and promote apoptosis of smooth muscle cells of penile corpus cavernosum.Upregulation of mitochondrial ATPase can inhibit the TGF-β1 pathway,improve the degree of fibrosis of penile cavernous smooth muscle,restore the function of smooth muscle,and improve the penile erectile function.