图1 各组大鼠阴茎海绵体HE染色(×200)
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Objective: To explore the mechanism of Yougui Pill in the treatment of DED by observing the effect of Yougui Pill on oxidative stress in corpus cavernosum tissue of diabetic erectile dysfunction (DED) rats.
The diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ) in mature male SD rats. The erectile function of rats was observed by apomorphine (APO) test and the DED model was screened. DED rats were divided into model group (n=10) and Yougui Pill group (n=10), and 10 of the most robust male SD rats were selected as control group. Eight weeks after administration, the erection of rats was observed by apomorphine test, the blood of tail vein was taken to determine the level of glucose. The corpus cavernosum tissue of rats were taken, of which the concentration of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and glutathione reductase (GSH-Px) were determined, and the apoptosis of endothelial cells were determined by TUNEL staining. The protein expression levels of nuclear erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were measured by Western blot.
①Compared with the control group, the blood glucose level increased, the erectile function decreased, the antioxidant capacity decreased, and the cell apoptosis increased in the model group. ②Compared with the model group, in Yougui Pill group, the total number of erections increased, the erectile latent time decreased, and the blood glucose level had no significant difference, but the content of MDA decreased significantly (P<0.05), the activity of SOD and GSH-Px as well as the expression of Nrf2 and HO-1 protein increased significantly (P<0.05), and the cell apoptosis decreased.
Yougui Pill may reduce oxidative stress injury and apoptosis of corpus cavernosum endothelial cells in DED rats by activating Nrf2-ARE signal pathway, thus improving diabetic erectile dysfunction.
糖尿病是一种常见的慢性疾病,由其引起的勃起功能障碍是糖尿病的慢性并发症之一[
1.1.1 动物
健康雄性SD大鼠,清洁级,体质量180~220 g,购自凯学生物科技(上海)有限公司,动物证号:SYXK(沪)2020-0003。所有动物饲养于上海中医药大学动物实验中心,实验室温度25~28 ℃,相对湿度65%~70%,光照周期:白天12 h/夜晚12 h,自由饮食饮水。研究前进行交配试验证实所有大鼠的性功能正常。
1.1.2 药物与试剂
右归丸(浓缩丸,批准文号:Z20201030),北京同仁堂股份有限公司同仁堂制药厂。右归丸(大蜜丸,每丸9 g,批号:Z20201030),购自上海中医药大学附属曙光医院中成药房,其组成及相应药物含量:熟地黄1.8 g,鹿角胶0.9 g、菟丝子0.9 g、山药0.9 g、枸杞子0.9 g、盐杜仲0.9 g,酒萸肉0.675 g、当归0.675 g,炮附子0.45 g、肉桂0.45 g。右归丸混悬液的制备:将右归丸完全溶解于双蒸水中,参照成人的体质量及计量,根据《药理实验方法学》[
链脲佐菌素(STZ,货号:S17049)、阿扑吗啡(APO,货号:A7247290)、TUNEL检测试剂盒(货号:C0002),美国Sigma公司;丙二醛(MDA,货号:BC0020)、超氧化物歧化酶(SOD,货号:QS1500)和谷胱甘肽还原酶(GSH-Px,货号:SG3664)试剂盒,南京建成生物工程研究所;二喹啉酸蛋白试剂盒(货号:CEK552Ge)、β-actin抗体(货号:A010102),炎熙生物科技有限公司;核因子E2相关因子(Nrf2,货号:PA561181)、血红素加氧酶-1(HO-1,货号:PE9376582),美国Santa Cruz Biotechnology公司;羊抗鼠IgG二抗(货号:SF13281),上海容创生物科技有限公司。STZ溶液的配制:STZ溶解于0.1 mmol/L柠檬酸-柠檬酸钠缓冲液(pH=4.5)中,浓度为20 mg/mL,注意避光,现用现配。APO溶液的配制:APO溶解于0.5 mg/kg的维生素C与生理盐水中,调整体积为5 ml/kg。
1.1.3 仪器
SimpliAmp型聚合酶链式反应扩增仪、高速冷冻离心机,美国ABI公司;PowerTome-PC型切片机,美国RMC公司;MaxQ8000型脱色摇床、Multiskan FC型酶标仪,美国 Thermo labsystem公司;ACCU-CHEK型简易血糖仪,美国ROCHE公司。
按随机数字表法从所有大鼠中选出10只大鼠作为正常对照组,其余大鼠进行造模。参照文献方法[
分组结束后6 h即开始干预治疗。右归丸组灌胃给予右归丸混悬液干预,连续灌胃8周,1次/d,2 ml/(200 g·次)。正常对照组与模型组灌胃给予等剂量双蒸水。
1.4.1 血糖测定
治疗结束后,各组大鼠称重,尾静脉取血,用血糖试纸测定血糖值。
1.4.2 勃起功能评估
大鼠颈部皮下注射APO(100 μg/kg),观察30 min,记录阴茎有无勃起、勃起次数及勃起潜伏期。
1.4.3 氧化应激指标测定
3%戊巴比妥钠以 0.2 ml/kg 腹腔注射麻醉大鼠,剥离大鼠阴茎海绵体组织,对标本进行常规的冰冻、OCT包埋、切片处理。HE染色观察海绵体组织血管分布、海绵窦等情况。取其中一部分阴茎海绵体组织制成匀浆后离心,取上清液,硫代巴比妥酸法检测MDA含量,比色法测定GSH-Px活力、黄嘌呤氧化酶法检测SOD活力。
1.4.4 TUNEL染色检测细胞凋亡
取各组大鼠组织石蜡切片,二甲苯、梯度乙醇洗脱后,磷酸盐缓冲液(PBS)冲洗,置于含2%蛋白激酶K的超纯水中室温孵育15 min,超纯水洗涤2次。加入含有vWF(内皮细胞标注物)的TUNEL反应液,37 ℃避光孵育1 h,PBS冲洗2次,加入4',6-二脒基-2-苯基吲哚(DAPI)、20%甘油缓冲液封片。置于荧光显微镜下观察,以激发波长552 nm、发射波长535 nm下显红色荧光细胞为凋亡细胞。
1.4.5 Nrf2、HO-1蛋白表达测定
采用Western blot法测定大鼠海绵体组织Nrf2、HO-1蛋白表达水平。
采用SPSS 19.0统计软件进行数据处理,计量资料以ˉx±s表示,多组间比较采用单因素方差分析,以P<0.05为差异有统计学意义。
如
组 别 | n | 血糖(nmol/L) | ||
---|---|---|---|---|
造模前 | 8周后 | 16周后 | ||
对照组 | 10 | 4.92±0.04 | 4.93±0.03 | 4.92±0.03 |
模型组 | 10 | 4.91±0.03 | 22.14±1.07* | 22.87±1.75* |
右归丸组 | 10 | 4.92±0.05 | 22.34±1.06 | 22.14±1.07 |
注: 与对照组比较,* P<0.05
如
组 别 | n | 阴茎勃起例数(例) | 勃起总次数(次) | 勃起平均潜伏时间(min) |
---|---|---|---|---|
空白对照组 | 10 | 10 | 24 | 11.8 |
模型组 | 10 | 1 | 1 | 29.6 |
右归丸组 | 10 | 6 | 6 | 23.6 |
注: 如无勃起,潜伏时间按30 min计算
阴茎海绵体HE染色可见对照组大鼠丰富的小血管及分布均匀的血窦,窦内可见部分红细胞,血窦内壁覆以扁平的内皮细胞(见
图1 各组大鼠阴茎海绵体HE染色(×200)
比较了大鼠抗氧化能力,与对照组比较,模型组MDA水平明显升高(P<0.05),SOD、GSH-Px水平明显下降(P<0.05);与模型组比较,右归丸组MDA水平明显下降(P<0.05),SOD、GSH-Px水平明显升高(P<0.05)。见
组 别 | 例数 | MDA (mmol/L) | SOD(ng/ml) | GSH-Px (ng/ml) |
---|---|---|---|---|
对照组 | 10 | 5.08±0.47 | 607.02±34.87 | 227.48±26.51 |
模型组 | 10 | 16.02±0.91* | 355.30±55.46* | 87.36±5.69* |
右归丸组 | 10 | 9.30±0.70# | 505.32±24.09# | 146.87±23.18# |
注: 与对照组比较,*P<0.05;与模型组比较,#P<0.05
Tunel+vWF荧光双染检测内皮细胞凋亡水平(见
图2 各组大鼠海绵体内皮细胞凋亡情况
如
图3 大鼠阴茎海绵体Nrf2、HO-1蛋白表达情况
组 别 | n | Nrf2 | HO-1 |
---|---|---|---|
空白组 | 10 | 1.36±0.16 | 0.85±0.05 |
模型组 | 10 | 0.35±0.05* | 0.18±0.03* |
右归丸组 | 10 | 0.75±0.05# | 0.37±0.03# |
注: 与对照组比较,*P<0.05;与模型组比较,#P<0.05
勃起功能障碍是当今社会一个重要的公共卫生问题,严重影响患者的生活质量。而患有糖尿病的男性发生勃起功能障碍的可能性是没有糖尿病的男性的三倍[
右归丸是“阴中求阳”的代表方,治疗DED疗效确切。我们前期的临床研究发现,右归丸可以明显改善DED患者的勃起功能,改善患者中医证候评分,使患者生活质量评分明显提高[
本研究观察到在用右归丸治疗8周后,DED大鼠的勃起功能有所改善。HE染色发现DED组大鼠海绵体血管结构紊乱,经右归丸治疗后的DED大鼠部分恢复海绵体血管结构。TUNEL染色发现,右归丸组大鼠海绵体内皮细胞凋亡数量较模型组有所减少。同时DED大鼠阴茎海绵体组织MAD水平升高,SOD、GSH-Px含量下降,说明右归丸组大鼠阴茎海绵体组织中抗氧化酶的含量均有不同程度的改善。由此可见,右归丸对DED大鼠勃起功能的改善可能是由于其增强了海绵体组织抗氧化能力,减轻了海绵体组织氧化应激损伤,并减少了海绵体血管内皮细胞的凋亡,进而达到改善大鼠勃起功能的作用,与相关研究结果[
综上所述,氧化应激损伤是DED重要的发病机制之一,右归丸可能通过激活Nrf2-ARE信号通路发挥抗氧化应激的作用,减少海绵体内皮细胞凋亡,进而改善勃起功能。同时,基于虑中医药对机体作用的多靶点和多途径性,右归丸整体调节机制仍处于不断探索中,有待进一步深入研究。
KOUIDRAT Y,PIZZOL D,COSCO T,et al.High prevalence of erectile dysfunction in diabetes: a systematic review and meta-analysis of 145 studies[J].Diabet Med, 2017, 34(9): 1185-1192. [Baidu Scholar]
YU X D,WANG J S,ZUO G,et al.Traditional Chinese medicine on treating diabetic mellitus erectile dysfunction: Protocol for a systematic review and meta-analysis[J].Medicine (Baltimore),2019, 98(13): e14928. [Baidu Scholar]
GUL M,SEREFOGLU E C. An update on the drug safety of treating erectile dysfunction[J]. Expert Opin Drug Saf, 2019, 18(10): 965-975. [Baidu Scholar]
HE J,LI X,DAI H H,et al.The safety and efficacy of PDE5-inhibitors-vardenafil on treating diabetes mellitus erectile dysfunction: A protocol for systematic review and meta analysis[J]. Medicine(Baltimore), 2019, 98(51): e18361. [Baidu Scholar]
魏伟,吴希美,李元建.药理实验学方法[M].北京:人民卫生出版社,2010: 1032. [Baidu Scholar]
KIM S W,ZHU G Q, BAE W J. Mesenchymal Stem Cells Treatment for Erectile Dysfunction in Diabetic Rats[J]. Sex Med Rev, 2020, 8(1): 114-121. [Baidu Scholar]
JAEGER V K, WALKER U A.Walker.Erectile dysfunction in systemic sclerosis[J]. Curr Rheumatol Rep, 2016, 18: 49. [Baidu Scholar]
THORVE V S,KSHIRSAGAR A D,VYAWAHARE NS, et al.Diabetes-induced erectile dysfunction: epidemiology, pathophysiology and management[J].J Diabetes Complications,2011, 25: 129-136. [Baidu Scholar]
ZHANG K Q,CHEN D,SUN D Q,et al.Probucol improves erectile function by restoring endothelial function and preventing cavernous fibrosis in streptozotocin-induced diabetic rats[J]. Urology, 2016, 91: 241.e9-241.e16. [Baidu Scholar]
LIU C,LU K,TAO T,et al.Endothelial nitric oxide synthase polymorphisms and erectile dysfunction: a meta-analysis[J]. J Sex Med, 2015, 12: 1319-1328. [Baidu Scholar]
ABDEL AZIZ M T, MOTAWI T, REZQ A,et al.Effects of a watersoluble curcumin protein conjugate vs. pure curcumin in a diabetic model of erectile dysfunction[J]. J Sex Med, 2012, 9: 1815-1833. [Baidu Scholar]
JOHNSON J M, BIVALACQUA T J, LAGODA G A, et al.eNOSuncoupling in age-related erectile dysfunction[J]. Int J Impot Res, 2011, 23: 43-48. [Baidu Scholar]
HUR W, GRAY N S. Small molecule modulators of antioxidant response pathway[J]. Curr Opin Chem Biol, 2011, 15: 162-173. [Baidu Scholar]
DU Y, ZHANG X, JI H, et al.Probucol and atorvastatin in combination protect rat brains in MCAO model: upregulating Peroxiredoxin2, Foxo3a and Nrf2 expression[J]. Neurosci Lett, 2012, 509: 110-115. [Baidu Scholar]
LIU L H, GUO Z,FENG M, et al.Protection of DDAH2 overexpression against homocysteine-induced impairments of DDAH/ADMA/NOS/NO pathway in endothelial cells[J]. Cell Physiol Biochem, 2012, 30: 1413-1422. [Baidu Scholar]
翟新宇,葛旻垚.右归丸治疗肾阴阳两虚型糖尿病性勃起功能障碍的临床研究[J].上海中医药杂志,2020, 54(S1): 61-63. [Baidu Scholar]
翟新宇,谈鸣岳,葛旻垚,等.基于网络药理学分析右归丸治疗糖尿病性勃起功能障碍的作用机制[J].中国男科学杂志,2021, 35(3): 32-39. [Baidu Scholar]
刘晓强,吕峥,孙光,等.右归胶囊改善糖尿病大鼠勃起功能障碍机制的实验研究[J].世界中医药,2012, 7(5): 445-447. [Baidu Scholar]
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